Interleukin-13 receptor alpha 2 peptide-based brain cancer vaccines

ABSTRACT

Provided herein are interleukin-13 receptor α2 peptide-based brain cancer vaccines and methods for treating and vaccinating against brain cancer comprising administering to patients in need thereof interleukin-13 receptor α2 peptide-based brain cancer vaccines. Also provided herein are regimens comprising interleukin-13 receptor α2 peptides and at least one additional peptide and/or immunostimulant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 14/724,127, filed May 28, 2015, which is acontinuation of U.S. patent application Ser. No. 14/515,939, filed Oct.16, 2014, which is a continuation of U.S. patent application Ser. No.13/925,093, filed Jun. 24, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/215,938, filed Aug. 23, 2011, which claims thebenefit of U.S. Provisional Patent Application No. 61/376,582, filedAug. 24, 2010 and which is a continuation-in-part of U.S. patentapplication Ser. No. 12/561,973, filed Sep. 17, 2009, which issued asU.S. Pat. No. 8,859,488 and which is a continuation of U.S. patentapplication Ser. No. 11/231,618, filed Sep. 21, 2005, which issued asU.S. Pat. No. 7,612,162 and which claims the benefit of U.S. ProvisionalPatent Application No. 60/611,797, filed Sep. 21, 2004. Each of theseapplications is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant NumbersNS040923, CA133859 and CA117152 awarded by the National Institutes ofHealth. The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 3,324 Kilobyte ASCII (Text) file named“751349_ST25.TXT,” dated Dec. 1, 2020.

1. INTRODUCTION

Provided herein are interleukin-13 receptor α2 peptide-based braincancer vaccines and methods for treating and vaccinating against braincancer comprising administering to patients in need thereofinterleukin-13 receptor α2 peptide-based brain cancer vaccines. Alsoprovided herein are vaccine regimens comprising interleukin-13 receptorα2 peptides and at least one additional peptide and/or immunostimulant.

2. BACKGROUND

Brain tumors are particularly difficult to treat using conventionalmethods such as surgery, radiotherapy, or chemotherapy. Factors such asinvasive growth patterns and the blood-brain barrier make the treatmentof malignant gliomas more problematic than other tumors. The lack ofeffective treatment options for patients has led to the development ofalternative therapies, such as immunotherapy.

Immunotherapy is a promising new approach in the treatment of malignantgliomas. The efficacy of peripheral immunizations with autologous gliomacells or dendritic cells (DC) pulsed with synthetic peptides fortumor-antigen-specific T cell epitopes has been demonstrated inpreclinical mouse models (Okada et al., 2001; Okada et al., 1998).Specific T cell epitope-based vaccines are likely safer than wholeglioma cell-based vaccines due to the lack of theoretical autoimmuneresponses against normal brain components. Such antigen-specificapproaches may also be more effective than the bulk tumor-antigenapproaches because presentation of immunogenic T cell-epitopes andstimulation of antigen-specific T cell precursors can take place moreefficiently with the use of specific antigen-peptides than bulk tumorantigens.

The identification of T cell immuno-epitopes in human glioma associatedantigens is required for the development of such vaccines against humangliomas. Few cytotoxic T lymphocyte (CTL) immuno-epitopes have beenidentified for human malignant gliomas. However, an HLA (human leukocyteantigen)-A2-restricted cytotoxic T lymphocyte (CTL) epitope derived fromthe interleukin (IL)-13 receptor (R) α2 was recently identified (Okanoet al., 2002). IL-13Rα2 is known to be expressed in the majority ofhuman malignant gliomas but not in normal tissues (Debinski et al.,2000), thus making the identified epitope (IL-13Rα2₃₄₅₋₃₅₃) anattractive component of peptide-based vaccines for gliomas. Bygenerating unique CTL lines by stimulation of CD8+ cells with thepeptide IL-13Rα2₃₄₅₋₃₅₃, it was demonstrated that IL-13Rα2 positive,HLA-A2 positive glioma cells were efficiently lysed in anantigen-specific manner. However, it remains unclear how efficientlysuch peptide-based vaccines can induce specific CTLs and whetherpeptide-analogues can be used for optimal expansion and activation ofIL-13Rα2 specific HLA-A2-restricted CTL.

It has been demonstrated that certain amino acid substitutions inpeptides identified as CTL epitopes could greatly enhance the bindingaffinity of such peptides to the HLA (human leukocyte antigen) complexand thus would augment the immunogenicity of the peptide (Bownds et al.,2001; Chen et al., 2000). The enhancement of the immunogenicity ofIL-13Rα2₃₄₅₋₃₅₃, and other such epitopes could lead to the developmentof powerful, tumor-specific peptide-based vaccines, which would be asignificant improvement in the current treatment regime for malignantgliomas. However, there remains a need for an improved polypeptideHLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope.

As discussed above, few cytotoxic T lymphocyte (CTL) immuno-epitopeshave been identified for human malignant gliomas. Given the markedantigenic heterogeneity of gliomas, however, immunotherapy with a singletumor-specific T-cell epitope might merely promote transientstabilization of disease, prior to the progression of antigen lossvariants. EphA2 is a member of the Eph family of receptor tyrosinekinases, comprised of two major classes (EphA2 and EphB), which aredistinguished by their specificities for ligands (ephrin-A and ephrin-B,respectively). EphA2 is frequently overexpressed and often functionallydysregulated in advanced cancers, such as metastatic lesions (Kinch etal., 2003). Due to the aggressive and invasive nature of malignantgliomas, EphA2 might be expressed in this tumor entity and could be apotential target for glioma vaccines. T-cell immunoepitopes in EphA2have been identified and characterized as potential targets andsurrogate markers for other forms of cancer immunotherapy (Alves et al.,2003, and Tatsumi et al., 2003). The identification of additional CTLepitopes is a necessary step in the development of multiepitope-basedvaccines for glioma which would be a significant improvement in thecurrent treatment regime for malignant gliomas.

3. SUMMARY

In one aspect, provided herein is a peptide derived from IL-13Rα2, whichserves as a HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. TheIL-13Rα2 peptide can comprise, consist of, or consist essentially of asubstitution mutant variant of WLPFGFILI (SEQ ID NO:1), wherein at leastone of the amino acid residues can be substituted for an amino acidother than the indicated residue. In addition, the IL-13Rα2 peptide cancomprise, consist of, or consist essentially of any of the followingsequences: WLPFGFILV (SEQ ID NO:2), ALPFGFILV (SEQ ID NO:3), orELPFGFILV (SEQ ID NO:4).

Also provided herein is a use of any of the above IL-13Rα2 peptides as avaccine for glioma. In addition, the invention provides a method ofvaccinating a patient against glioma, where the peptide is introducedinto a patient under conditions sufficient for the patient to develop aCTL response. Further, provided herein is a use of an EphA2 peptidehaving the sequence TLADFDPRV (SEQ ID NO:6) or a composition comprisingsaid peptide and a physiologically acceptable carrier, as a vaccine forglioma. Also provided herein is a method of vaccinating a patientagainst glioma, wherein an EphA2 peptide having the sequence TLADFDPRV(SEQ ID NO:6) or a composition comprising said peptide and aphysiologically acceptable carrier, is introduced into a patient underconditions sufficient for the patient to develop a CTL response.

In another aspect, presented herein are IL-13Rα2 peptide-based vaccinescomprising an IL-13Rα2 peptide and one, two, three, or more additionalbrain cancer-associated peptides. In certain embodiments, the IL-13Rα2peptide-based vaccines described herein are administered concurrentlywith one or more helper T cell epitopes and/or one or more immuneresponse modifiers. In accordance with such embodiments, the one or morehelper T cell epitopes and/or one or more immune response modifiers maybe administered as part of the vaccine (e.g., in solution with theIL-13Rα2 peptide and the one, two, three, or more additional braincancer-associated peptides) or separate from the vaccine (i.e., thehelper T cell epitopes and/or immune response modifiers may beadministered as a formulation that is not a part of the vaccineformulation). In some embodiments, the IL-13Rα2 peptide-based vaccinesdescribed herein are administered as cell-free vaccines. In anotherembodiment, the IL-13Rα2 peptide-based vaccine is administered with anadjuvant. In a preferred embodiment, the IL-13Rα2 peptide-based vaccineis administered in combination with additional peptides. In anotherembodiment, the IL-13Rα2 peptide-based vaccine is administered with animmunomodulatory agent. —In another embodiment, the IL-13Rα2peptide-based vaccine is administered as an emulsion in Montanide ISA51, as a component of a regimen that includes injections with animmunostimulatory agent. In a preferred embodiment, theimmunostimulatory agent is poly ICLC. In other embodiments, the IL-13Rα2peptide-based vaccines described herein are administered as dendriticcell vaccines.

In one embodiment, an IL-13Rα2 peptide-based vaccine comprises anIL-13Rα2 peptide, an EphA2 peptide, a YKL-40 peptide, and a GP100peptide. In a specific embodiment, an IL-13Rα2 peptide-based vaccinecomprises the IL-13Rα2 peptide corresponding to any one of SEQ IDNOs:1-4, the EphA2 peptide corresponding to SEQ ID NO:6, the YKL-40peptide corresponding to SEQ ID NO:10, and the GP100 peptidecorresponding to SEQ ID NO:11. In another specific embodiment, anIL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptidecorresponding to SEQ ID NO:3, the EphA2 peptide corresponding to SEQ IDNO:6, the YKL-40 peptide corresponding to SEQ ID NO:10, and the GP100peptide corresponding to SEQ ID NO:11. In some embodiments, the IL-13Rα2peptide-based vaccine is administered concurrently with one or morehelper T cell epitopes. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with a helper T cellepitope, wherein the helper T cell epitope is the PADRE peptide. In someembodiments, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with one or more immune response modifiers. In someembodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine.In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendriticcell vaccine.

In another embodiment, an IL-13Rα2 peptide-based vaccine comprises anIL-13Rα2 peptide, an EphA2 peptide, a survivin peptide, and a WT1peptide. In a specific embodiment, an IL-13Rα2 peptide-based vaccinecomprises the IL-13Rα2 peptide corresponding to any one of SEQ IDNOs:1-4, the EphA2 peptide corresponding to SEQ ID NO:6, the survivinpeptide corresponding to SEQ ID NO:7, and the WT1 peptide correspondingto SEQ ID NO:8. In another specific embodiment, an IL-13Rα2peptide-based vaccine comprises the IL-13Rα2 peptide corresponding toSEQ ID NO:3, the EphA2 peptide corresponding to SEQ ID NO:6, thesurvivin peptide corresponding to SEQ ID NO:7, and the WT1 peptidecorresponding to SEQ ID NO:8. In some embodiments, the IL-13Rα2peptide-based vaccine is administered concurrently with one or morehelper T cell epitopes. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with a helper T cellepitope, wherein the helper T cell epitope is the Tetanus toxoid. Insome embodiments, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with one or more immune response modifiers. In a specificembodiment, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with an immune response modifier, wherein the immuneresponse modifier is poly-ICLC. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with an immuneresponse modifier, wherein the immune response modifier is MontanideISA-51. In some embodiments, the IL-13Rα2 peptide-based vaccine is acell-free vaccine. In other embodiments, the IL-13Rα2 peptide-basedvaccine is a dendritic cell vaccine.

In another embodiment, an IL-13Rα2 peptide-based vaccine comprises anIL-13Rα2 peptide, an EphA2 peptide, and a survivin peptide. In aspecific embodiment, an IL-13Rα2 peptide-based vaccine comprises theIL-13Rα2 peptide corresponding to any one of SEQ ID NOs:1-4, the EphA2peptide corresponding to SEQ ID NO:6, and the survivin peptidecorresponding to SEQ ID NO:7. In another specific embodiment, anIL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptidecorresponding to SEQ ID NO:3, the EphA2 peptide corresponding to SEQ IDNO:6, and the survivin peptide corresponding to SEQ ID NO:7. In someembodiments, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with one or more helper T cell epitopes. In a specificembodiment, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with a helper T cell epitope, wherein the helper T cellepitope is the Tetanus toxoid. In some embodiments, the IL-13Rα2peptide-based vaccine is administered concurrently with one or moreimmune response modifiers. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with an immuneresponse modifier, wherein the immune response modifier is poly-ICLC. Ina specific embodiment, the IL-13Rα2 peptide-based vaccine isadministered concurrently with an immune response modifier, wherein theimmune response modifier is Montanide ISA-51. In some embodiments, theIL-13Rα2 peptide-based vaccine is a cell-free vaccine. In otherembodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cellvaccine.

4. DEFINITIONS

As used herein, the terms “about” or “approximately” when used inconjunction with a number refers to any number within 1, 5 or 10% of thereferenced number.

As used herein, the term “agent” refers to any molecule, compound,and/or substance that can be used in or in combination with aninterleukin-13 receptor α2 peptide-based brain cancer vaccines describedherein. The term agent includes, without limitation, proteins,immunoglobulins (e.g., multi-specific Igs, single chain Igs, Igfragments, polyclonal antibodies and their fragments, monoclonalantibodies and their fragments), peptides (e.g., peptide receptors,selectins), binding proteins, biologics, chemospecific agents,chemotoxic agents, anti-angiogenic agents, and small molecule drugs.

As used herein, the term “amino acid sequence identity” refers to thedegree of identity or similarity between a pair of aligned amino acidsequences, usually expressed as a percentage. As used herein, the terms“percent identity,” “percent identical,” “% identity,” and “% identical”with respect to amino acid sequence refer to the percentage of aminoacid residues in a candidate sequence that are identical (i.e., theamino acid residues at a given position in the alignment are the sameresidue) to the corresponding amino acid residue in the peptide afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence homology. As used herein, the terms“percent similarity,” “percent similar,” “% similarity,” and “% similar”with respect to amino acid sequence refer to the percentage of aminoacid residues in a candidate sequence that are similar (i.e., the aminoacid substitution at a given position in the alignment is a conservativesubstitution, as discussed below), to the corresponding amino acidresidue in the peptide after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence homology.Sequence homology, including percentages of sequence identity andsimilarity, are determined using sequence alignment techniqueswell-known in the art, including computer algorithms designed for thispurpose, using the default parameters of said computer algorithms or thesoftware packages containing them.

As used herein, the term “conservative substitution” refers toreplacement of an amino acid of one class with another amino acid of thesame class. In particular embodiments, a conservative substitution doesnot alter the structure or function, or both, of a peptide. Classes ofamino acids for the purposes of conservative substitution includehydrophobic (Met, Ala, Val, Leu, Ile), neutral hydrophilic (Cys, Ser,Thr), acidic (Asp, Glu), basic (Asn, Gln, His, Lys, Arg), conformationdisrupters (Gly, Pro) and aromatic (Trp, Tyr, Phe).

As used herein, the term “peptide” refers to a polymer of amino acidslinked by amide bonds as is known to those of skill in the art. Apeptide can be a polymer of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acidslinked by covalent amide bonds. In some embodiments, the peptide is apolymer of 6 to 8, 8 to 10, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10to 40, 10 to 50, or 25 to 25 amino acids linked by covalent amide bonds.In certain embodiments, the peptide is a polymer of 50 to 65, 50 to 75,50 to 85, 50 to 95, 50 to 100, 75 to 100 amino acids linked by covalentamide bonds. As used herein, the term can refer to a single peptidechain linked by covalent amide bonds. The term can also refer tomultiple peptide chains associated by non-covalent interactions such asionic contacts, hydrogen bonds, Van der Waals contacts and hydrophobiccontacts. Those of skill in the art will recognize that the termincludes peptides that have been modified, for example bypost-translational processing such as signal peptide cleavage, disulfidebond formation, glycosylation (e.g., N-linked glycosylation), proteasecleavage and lipid modification (e.g. S-palmitoylation).

As used herein, the terms “purified” and “isolated” when used in thecontext of a peptide that is obtained from a natural source, e.g.,cells, refers to a peptide which is substantially free of contaminatingmaterials from the natural source, e.g., soil particles, minerals,chemicals from the environment, and/or cellular materials from thenatural source, such as but not limited to cell debris, cell wallmaterials, membranes, organelles, the bulk of the nucleic acids,carbohydrates, proteins, and/or lipids present in cells. Thus, a peptidethat is isolated includes preparations of a polypeptide having less thanabout 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular materialsand/or contaminating materials. As used herein, the terms “purified” and“isolated” when used in the context of a peptide that is chemicallysynthesized refers to a peptide which is substantially free of chemicalprecursors or other chemicals which are involved in the syntheses of thepolypeptide.

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid can be single-stranded or double-stranded.

As used herein, the phrase “prophylactic vaccine” refers to a vaccinedescribed herein that is used for the purpose of preventing cancer.

As used herein, the term “prophylactically effective regimen” refers toan effective regimen for dosing, timing, frequency and duration of theadministration of one or more therapies for the prevention of braincancer or a symptom thereof.

As used herein, the term “therapeutic vaccine” refers to a vaccinedescribed herein that is used for the purpose of treating and/ormanaging brain cancer.

As used herein, the term “therapeutically effective regimen” refers to aregimen for dosing, timing, frequency, and duration of theadministration of one or more therapies for the treatment and/ormanagement of brain cancer or a symptom thereof.

As used herein, the terms “subject” or “patient” are usedinterchangeably to refer to an animal (e.g., birds, reptiles, andmammals). In a specific embodiment, a subject is a bird. In anotherembodiment, a subject is a mammal including a non-primate (e.g., acamel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, andmouse) and a primate (e.g., a monkey, chimpanzee, and a human). Incertain embodiments, a subject is a non-human animal. In someembodiments, a subject is a farm animal or pet. In another embodiment, asubject is a human. In another embodiment, a subject is a human infant.In another embodiment, a subject is a human toddler. In anotherembodiment, a subject is a human child. In another embodiment, a subjectis a human adult. In another embodiment, a subject is an elderly human.

As used herein, the term “human infant” refers to a newborn to 1 yearold human.

As used herein, the term “human toddler” refers to a human that is 1years to 3 years old.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “elderly human” refers to a human 65 years orolder.

As used herein, the term “brain cancer” refers to a tumor located insidethe cranium or in the central spinal canal. Brain cancer refers to bothprimary tumors (i.e., tumors that originate in the intracranial sphereor the central spinal canal) and secondary tumors (i.e., tumors thatinvaded the intracranial sphere or the central spinal canal afteroriginating from tumors primarily located in other organs).

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), composition(s), formulation(s), and/or agent(s)that can be used in the prevention or treatment of brain cancer or adisease or symptom associated therewith. In certain embodiments, theterms “therapies” and “therapy” refer to biological therapy, supportivetherapy, and/or other therapies useful in treatment or prevention ofbrain cancer or a disease or symptom associated therewith known to oneof skill in the art.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of brain cancer and/or one or moresymptoms thereof, to enhance or improve the prophylactic effect(s) ofanother therapy, reduce the severity, the duration of brain cancer,ameliorate one or more symptoms of brain cancer, prevent the advancementof brain cancer, cause regression of brain cancer, and/or enhance orimprove the therapeutic effect(s) of another therapy.

As used herein, the term “in combination” in the context of theadministration of a therapy to a subject refers to the use of more thanone therapy (e.g., prophylactic and/or therapeutic). The use of the term“in combination” does not restrict the order in which the therapies(e.g., a first and second therapy) are administered to a subject. Atherapy can be administered prior to (e.g., 1 minute, 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy to a subject which had, has, or is susceptible to braincancer. The therapies are administered to a subject in a sequence andwithin a time interval such that the therapies can act together. In aparticular embodiment, the therapies are administered to a subject in asequence and within a time interval such that they provide an increasedbenefit than if they were administered otherwise. Any additional therapycan be administered in any order with the other additional therapy.

As used herein, the terms “manage,” “managing,” and “management” in thecontext of the administration of a therapy to a subject refer to thebeneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic vaccine) or a combination of therapies,while not resulting in a cure of brain cancer. In certain embodiments, asubject is administered one or more therapies (e.g., one or moreprophylactic or therapeutic vaccines) to “manage” brain cancer so as toprevent the progression or worsening of the condition.

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy to a subject refer to theprevention or inhibition of the recurrence, onset, and/or development ofbrain cancer or a symptom thereof in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or a combination of therapies (e.g., a combination of prophylactic ortherapeutic agents).

As used herein, the term “concurrently” means sufficiently close in timeto produce a combined effect (that is, concurrently may besimultaneously, or it may be two or more events occurring within a timeperiod before or after each other). When administered with other agents,the IL-13Rα2 peptide-based vaccines provided herein may be administeredconcurrently with the other active agent. In some embodiments anIL-13Rα2 peptide-based vaccine provided herein and one or more otheragents (e.g., a helper T cell epitope, an adjuvant, and/or an immuneresponse modifier) are administered to a subject concurrently, whereinthe administration IL-13Rα2 peptide-based vaccine provided herein andone or more other agents are in the same composition. In otherembodiments an IL-13Rα2 peptide-based vaccine provided herein and one ormore other agents (e.g., a helper T cell epitope, an adjuvant, and/or animmune response modifier) are administered to a subject concurrently,wherein the administration IL-13Rα2 peptide-based vaccine providedherein and one or more other agents are not in the same composition. Inone embodiment, the agent that is administered concurrently with theIL13Rα2 peptide-based vaccine is administered as a separate injection.In certain embodiments, an IL-13Rα2 peptide-based vaccine providedherein and one or more other agents e.g., a helper T cell epitope, anadjuvant, and/or an immune response modifier) are administered to asubject concurrently, wherein the concurrent administration is separatedby at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or 2weeks.

As used herein, the term “brain cancer-associated peptide” refers to apeptide found to be associated with one or more brain cancers and whichserves as an HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitope. Insome embodiments, a brain cancer-associated peptide is aglioma-associated peptide, i.e., the brain cancer that the peptide isassociated with is glioma. In a preferred embodiment, the braincancer-associated peptide is expressed by glioma cells. Exemplary braincancer-associated peptides include, without limitation, IL-13Rα2peptides, EphA2 peptides, YKL-40 peptides, GP100 peptides, survivinpeptides, and WT1 peptides.

As used herein, the term “IL-13Rα2 peptide” refers to a peptide derivedfrom the IL-13Rα2 protein and which serves as an HLA-A2 restrictedcytotoxic T lymphocyte (CTL) epitope. In a specific embodiment theIL-13Rα2 protein from which an IL-13Rα2 peptide is derived is the humanIL-13Rα2 protein. In another specific embodiment, an IL-13Rα2 peptidecomprises any one of SEQ ID NOs:1-4. In some embodiments, an IL-13Rα2peptide comprises one, two, three, or more amino acid mutations (e.g.,additions, substitutions, or deletions) relative to the IL-13Rα2 peptideas it exists in the native (e.g., wild-type) form of the IL-13Rα2protein.

As used herein, the term “EphA2 peptide” refers to a peptide derivedfrom the EphA2 protein and which serves as an HLA-A2 restrictedcytotoxic T lymphocyte (CTL) epitope. In a specific embodiment the EphA2protein from which an EphA2 peptide is derived is the human EphA2protein. In another specific embodiment, an EphA2 peptide comprises SEQID NO:6. In some embodiments, an EphA2 peptide comprises one, two,three, or more amino acid mutations (e.g., additions, substitutions, ordeletions) relative to the EphA2 peptide as it exists in the native(e.g., wild-type) form of the EphA2 protein.

As used herein, the term “YKL-40 peptide” refers to a peptide derivedfrom the YKL-40 protein and which serves as an HLA-A2 restrictedcytotoxic T lymphocyte (CTL) epitope. In a specific embodiment theYKL-40 protein from which a YKL-40 peptide is derived is the humanYKL-40 protein. In another specific embodiment, a YKL-40 peptidecomprises SEQ ID NO:10. In some embodiments, a YKL-40 peptide comprisesone, two, three, or more amino acid mutations (e.g., additions,substitutions, or deletions) relative to the YKL-40 peptide as it existsin the native (e.g., wild-type) form of the YKL-40 protein.

As used herein, the term “GP100 peptide” refers to a peptide derivedfrom the GP100 protein and which serves as an HLA-A2 restrictedcytotoxic T lymphocyte (CTL) epitope. In a specific embodiment the GP100protein from which a GP100 peptide is derived is the human GP100protein. In another specific embodiment, a GP100 peptide comprises SEQID NO:11. In some embodiments, a GP100 peptide comprises one, two,three, or more amino acid mutations (e.g., additions, substitutions, ordeletions) relative to the GP100 peptide as it exists in the native(e.g., wild-type) form of the GP100 protein.

As used herein, the term “survivin peptide” refers to a peptide derivedfrom the survivin protein and which serves as an HLA-A2 restrictedcytotoxic T lymphocyte (CTL) epitope. In a specific embodiment thesurvivin protein from which a survivin peptide is derived is the humansurvivin protein. In another specific embodiment, a survivin peptidecomprises SEQ ID NO:7. In some embodiments, a survivin peptide comprisesone, two, three, or more amino acid mutations (e.g., additions,substitutions, or deletions) relative to the survivin peptide as itexists in the native (e.g., wild-type) form of the survivin protein.

As used herein, the term “WT1 peptide” refers to a peptide derived fromthe WT1 protein and which serves as an HLA-A2 restricted cytotoxic Tlymphocyte (CTL) epitope. In a specific embodiment the WT1 protein fromwhich a WT1 peptide is derived is the human WT1 protein. In anotherspecific embodiment, a WT1 peptide comprises SEQ ID NO:8. In someembodiments, a WT1 peptide comprises one, two, three, or more amino acidmutations (e.g., additions, substitutions, or deletions) relative to theWT1 peptide as it exists in the native (e.g., wild-type) form of the WT1protein.

As used herein, the term “cell-free vaccine” refers to a vaccinecomprising an IL-13Rα2 peptide, wherein the IL-13Rα2 peptide is notloaded on a cell (e.g., a dendritic cell) in the vaccine (e.g., thepeptide derived from IL-13Rα2 is in solution). In a preferredembodiment, the peptides are emulsified in adjuvant. In anotherpreferred embodiment, the adjuvant is Montanide ISA 51.

As used herein, the term “dendritic cell vaccine” refers to a vaccinecomprising an IL-13Rα2 peptide, wherein the IL-13Rα2 peptide is loadedon dendritic cells in the vaccine.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically presents data demonstrating that IL-13Rα2-V9 andIL-13Rα2-A 1V9 induced a higher magnitude of CTL reactivity than thenative IL-13Rα2₃₄₅₋₃₅₃ or IL-13Rα2-E 1V9 against T2 cells loaded withvarious concentrations of native IL-13Rα2₃₄₅₋₃₅₃. CD8+ T cells from anHLA-A2+ glioma patient were stimulated with DCs loaded with eithernative IL-13Rα2₃₄₅₋₃₅₃ (●), IL-13Rα2-V9 (◯), IL-13Rα2-A1V9 (Δ),IL-13Rα2-E1V9 (X), Influenza M1₅₈₋₆₆ peptide (▾), or no peptide (□) for10 days. Then, the T cells were tested for lytic activity against T2cells loaded with indicated concentrations of IL-13Rα2₃₄₅₋₃₅₃ or nopeptide by 4-hr ⁵¹Cr-release assay. The E/T ratio was 12.5. P<0.01 forIL-13Rα2-V9 vs. native as well as IL-13Rα2-A 1V9 vs. native at 0.1 and 1nM by two-tailed Student-t test. These data demonstrate results from oneof three separate experiments with similar results.

FIG. 2 graphically presents data demonstrating that the CTL line inducedby the V9 peptide (open circles) had increased lytic activity against T2cells loaded with various concentrations of the wild typeIL-13Rα2₃₄₅₋₃₅₃ peptide. The CTL lines induced by each of the 3 agonistanalogues (V9 (open circles), A1V9 (triangles); E1V9 (X)) or the wildtype peptide (closed circles) were examined for CTL activities againstlower concentrations of target IL-13Rα2₃₄₅₋₃₅₃ peptide with T2 cellsloaded with various concentrations (1-100 nM) of IL-13Rα2₃₄₅₋₃₅₃ by 4-Hr⁵¹Cr-release assay (E/T ratio=50).

FIG. 3 graphically presents data demonstrating that the modifiedpeptides induced a higher magnitude of CTL reactivity than the nativeIL-13Rα2₃₄₅₋₃₅₃ against human glioma cell lines. CD8+ cells derived froman HLA-A2+ glioma patient were stimulated with native IL-13Rα2₃₄₅₋₃₅₃(●), IL-13Rα2-V9 (◯), IL-13Rα2-A1V9 (Δ), or IL-13Rα2-E1V9 (X). On day10, the cells were tested for lytic ability against human glioma cellsSNB19 (A) and U-251 (B) (both are IL-13Rα+/HLA-A2+) using 4-Hr⁵¹Cr-release assay. Against SNB19 glioma cells, p<0.05 at all E/T ratiosfor IL-13Rα2-V9 vs. native IL-13Rα2₃₄₅₋₃₅₃ as well as IL-13Rα2-A1V9 vs.native IL-13Rα2₃₄₅₋₃₅₃ by two-tailed Student-t tests. Against U251glioma cells, p<0.05 at E/T ratio of 10 and 40 for IL-13Rα2-V9 vs.native IL-13Rα2₃₄₅₋₃₅₃ as well as IL-13Rα2-A1V9 vs. nativeIL-13Rα2₃₄₅₋₃₅₃ by two-tailed Student-t tests. IL-13Rα2-E1V9 did notimprove the CTL reactivity for a statistically significant level incomparison to the native. The data presented represent one of threeexperiments with different donors with similar results.

FIG. 4 graphically presents data demonstrating that the addition of“cold” T2 cells pulsed with IL-13Rα2₃₄₅₋₃₅₃ inhibited the CTL activitiesindicating the antigen-specificity of the CTL lines. The CTL linesinduced with each peptide (control (A); Flu (B); IL-13Rα2₃₄₅₋₃₅₃ (C);IL-13Rα2_(345-9V) (D)) were incubated for 4 h with ⁵¹Cr-labeled humanglioma cell lines SNB19 at the indicated E:T ratios for evaluation ofspecific lytic ability (●). For the cold target inhibition assay,⁵¹Cr-labeled target SNB19 cells (1×10³ cells/well) and cold T2 cells(1×10⁴ cells/well) pulsed with (Δ) or without (◯) peptideIL-13Rα2₃₄₅₋₃₅₃ were incubated with the CTLs.

FIG. 5 graphically presents data demonstrating that the addition ofanti-HLA-A2 antibody inhibited the CTL activities indicatingHLA-A2-restricted recognition of the CTL lines. The CTL lines inducedwith each peptide (control (A); Flu (B); IL-13Rα2₃₄₅₋₃₅₃ (C);IL-13Rα2_(345-9V) (D)) were incubated for 4 h with ⁵¹Cr-labeled humanglioma cell line SNB19 at the indicated E:T ratios for evaluation ofspecific lytic ability (●). Anti-HLA-A2 antibody (W6/32; 10 μg/ml) wasadded to block the function of HLA-A2 mediated recognition by the Tcells (◯).

FIG. 6 graphically presents data demonstrating that the modifiedpeptides induced higher magnitude of CTL reactivity than the nativeIL-13Rα2₃₄₅₋₃₅₃ against EL4-HHD loaded with the native IL-13Rα2₃₄₅₋₃₅₃.SPCs obtained from HHD mice that had been immunized with either controlMART-127-35 (●), native IL-13Rα2₃₄₅₋₃₅₃ (◯), IL-13Rα2-V9 (Δ) orIL-13Rα2-A1V9 (X) were tested for their specific lytic activity againstEL4-HHD cells pulsed with the native IL-13Rα2₃₄₅₋₃₅₃ by standard 4 hr⁵¹Cr-release assays.

FIG. 7 graphically presents data demonstrating that the modifiedpeptides induced a higher magnitude of CTL reactivity than the nativeIL-13Rα2₃₄₅₋₃₅₃ against EL4-HHD-IL-13Rα2. SPCs obtained from HHD micethat had been immunized with either control MART-127-35 (A), nativeIL-13Rα2₃₄₅₋₃₅₃ (B), IL-13Rα2-V9 (C), or IL-13Rα2-A1V9 (D) were testedfor their specific lytic activity against EL4-HHD-IL-13Rα2 (◯) orcontrol EL4-HHD (●) by standard 4 hr ⁵¹Cr-release assays.

FIG. 8 depicts the expression of EphA2 protein in glioblastomamultiforme (GBM) and anaplastic astrocytoma (AA). Paraffin embeddedsections of surgical specimens obtained from patients with GBM (A-C) orAA (D) were deparaffinized and stained with anti-EphA2 polyclonalantibody (C-20: Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), orcontrol rabbit IgG (upper right corner window for each sample).Relatively dense staining on endothelia and tumor cells surrounding thevessel was observed (D). Nine of fourteen GBM and six of nine AA casesexamined were positive for EphA2 (not shown). Original magnification;×20.

FIG. 9 graphically presents data demonstrating that the CD8+ cellsstimulated with EphA2883-891 elicited CTL responses against human gliomacells expressing HLA-A2 and EphA2 protein. CD8+ T cells from an HLA-A2+glioma patients were stimulated with DCs loaded with EphA2883-891 for 10days. These T cells were then tested for their lytic activity againsthuman glioma cells SNB19 (HLA-A2+, EphA2+) (▴), U251 (HLA-A2+, EphA2+)(▪) and A172 (HLA-A2−, EphA2+) (▾) by 4-hr ⁵¹Cr-release assay.

FIG. 10: IL-12 production levels positively correlated with TTP.P=0.0255 based on Cox regression followed by likelihood-ratio test.Closed circles indicate patients who have already progressed, whereasclosed diamonds represent patients who have not recurred to date.

FIG. 11: T-cell responses to IL-13Rα2 (A), PADRE (B), EphA2 (C), YKL-40(D), or gp100 (E) evaluated by IFN-γ ELISPOT. Time course for IFN-γESLIPOT assays for all evaluated patients with box plots (boxes=25th to75th percentiles; vertical lines=minimum to maximum). Numbers at thebottom of each time point in the panel for YKL-40 (D) are the number ofassessable patients at the time shown. These numbers also pertain to theother GAAs and PADRE.

FIG. 12: T-cell responses to IL-13Rα2 (▪), PADRE (

), EphA2 (▴), YKL-40 (♦), or gp100 (●) evaluated by IFN-γ ESLIPOTanalyses for Patient 10.

FIG. 13: Patient 6 showed durable tetramer responses, which wereanalyzed for up to 33 weeks (IL-13Rα2 tetramer+cells (▪); EphA2tetramer+cells (▴); gp100 tetramer+cells (●)) (C). Examples of dot plotsfor positive tetramer responses against the IL-13Rα2-epitope are shown(A-B).

FIG. 14: Induction of type-1 cytokine and chemokine responses. Linegraphs represent paired relative gene expression of IFNα1 (A), CXCL10(B), CCL5 (C), IL-12α (D), TLR3 (E), or CCL22 (F) by RT-PCR on one dayprior to the 1^(st) vaccination compared to 24 hours post vaccine forcase number 10 (▪), 11(|), 16 (Δ), 19 (□), or 22 (♦). Y axes indicateconcentrations of cytokine/chemokines by pg/ml. Numbers in the panels ofeach of (A)-(F) indicate p-values based on paired student t test usingthe means of each patient's ΔΔC_(T) value.

FIG. 15: Induction of type-1 cytokine and chemokine responses. Linegraphs represent paired relative gene expression of IFNα1 (A), CXCL10(B), IFNγ (C), TLR3 (D) by RT-PCR on one day prior to the 1stvaccination compared to 9 weeks post 1^(st) vaccine for case number 9(●), 10 (▪), 12 (no symbol), 16 (Δ), 18 (□), 19 (▴), or 20 (1). Y axesindicate concentrations of cytokine/chemokines by pg/ml. Numbers in thepanels of each of (A)-(D) indicate p-values based on paired student ttest using the means of each patient's ΔΔC_(T) value.

FIG. 16: Luminex analyses were performed in pre-1^(st) and post-4^(th)vaccine serum samples. Y axes indicate concentrations ofcytokine/chemokines by pg/ml. Numbers in the panels of each of (A)-(E)indicate p-values based on paired student t test using the means of theconcentrations.

FIG. 17: Patient 1 demonstrated increase in the size of Gd-enhancedlesion following two booster vaccines and underwent surgical resectionof the lesion. In situ hybridization detected mRNA for CXCL10 (darkspots) in the post-vaccine tissue (B) but not in the initially resectedtumor (pre-vaccine) (A). Control, (C). None of two other pre-vaccinetissues demonstrated positive CXCL10 messages. The scale bar equals to100 μm. Hematoxylin and eosin staining was performed for background.

FIG. 18: Patients with clinical response. Patient 20 demonstratedcomplete radiological response of Gd-enhancing tumor on MRI on Weeks 17and 33 (three consecutive slices shown for Week 0 (A-C), Week 17 (D-F),and Week 33 (G-I)). Following two booster vaccines, Patient 1demonstrated enlargement of Gd-enhanced lesion. Resected tissue revealedno evidence of mitotically active tumor (J), but remarkable infiltrationof CD68⁺ macrophages (K) and CD8⁺ T-cells (L). Originalmagnifications×20 for J-L.

FIG. 19: Flow diagram for the trial. See Section 7.7 for details oftreatment. The second phase of booster vaccines could start any timeafter Week 37 and administered every 3 months up to 3 years from thefirst vaccine, unless patients demonstrated major AE or diseaseprogression. The αDC1 vaccines were administered using ultrasound toinguinal lymph nodes (right and left for the first and second vaccines,respectively) and axillary lymph nodes (right and left for the third andforth vaccines, respectively). The site was rotated in the same orderfor booster vaccines to minimize the potential effects ofinjection-induced trauma in the microenvironment of the lymph nodes byrepeating injections in short periods of time.

FIG. 20: Time to progression (A) and overall survival (B) for GBM (▪)and AG (♦). Median TTP are 4 and 13 months for GBM and AG, respectively.

FIG. 21: IFA-based peptide vaccines induce superior CTL activities toDC-based vaccines combined with intramuscular (i.m.) injections ofpoly-ICLC. C57BL/6 mice received two injections (on Days 0 and 7) ofeither: 1) subcutaneous (s.c.) ovalbumin₂₅₇₋₂₆₄ peptide emulsified inIFA (IFA-OVA) plus concurrent i.m. injection of poly-ICLC (50μg/injection); 2) s.c. IFA-OVA plus i.m. saline; 3) bone marrow-derivedDC loaded with ovalbumin₂₅₇₋₂₆₄ peptide (DC-OVA) plus i.m. poly-ICLC; or4) DC-OVA plus i.m. saline. Other control groups included mice receivingIFA or DC alone without the OVA-peptide. IFA-OVA vaccines combined withpoly-ICLC demonstrated the highest level of OVA-specific CTL in vivo.Use of non-mutated self GAA-peptides emulsified in IFA with poly-ICLCimproved survival of mice without inducing autoimmunity. These datademonstrate that poly-ICLC assisted IFA-peptide vaccines represent aneffective and safe vaccination strategy.

6. DETAILED DESCRIPTION

Provided herein are interleukin-13 receptor α2 (IL-13Rα2) peptide-basedvaccines comprising an IL-13Rα2 peptide. The IL-13Rα2 peptide-basedvaccines provided herein comprise an IL-13Rα2 peptide and at least oneadditional brain cancer-associated peptide.

In one aspect, presented herein are IL-13Rα2 peptide-based vaccinescomprising an IL-13Rα2 peptide and one, two, three, or more additionalbrain cancer-associated peptides. In certain embodiments, the IL-13Rα2peptide-based vaccines described herein are administered concurrentlywith one or more helper T cell epitopes and/or one or more immuneresponse modifiers. In accordance with such embodiments, the one or morehelper T cell epitopes and/or one or more immune response modifiers maybe administered as part of the vaccine (e.g., in solution with theIL-13Rα2 peptide and the one, two, three, or more additional braincancer-associated peptides) or separate from the vaccine (i.e., thehelper T cell epitopes and/or immune response modifiers may beadministered as a formulation that is not a part of the vaccineformulation). In some embodiments, the IL-13Rα2 peptide-based vaccinesdescribed herein are administered as cell-free vaccines. In otherembodiments, the IL-13Rα2 peptide-based vaccines described herein areadministered as dendritic cell vaccines.

In one embodiment, an IL-13Rα2 peptide-based vaccine comprises anIL-13Rα2 peptide, an EphA2 peptide, a YKL-40 peptide, and a GP100peptide. In a specific embodiment, an IL-13Rα2 peptide-based vaccinecomprises the IL-13Rα2 peptide corresponding to any one of SEQ IDNOs:1-4, the EphA2 peptide corresponding to SEQ ID NO:6, the YKL-40peptide corresponding to SEQ ID NO:10, and the GP100 peptidecorresponding to SEQ ID NO:11. In another specific embodiment, anIL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptidecorresponding to SEQ ID NO:3, the EphA2 peptide corresponding to SEQ IDNO:6, the YKL-40 peptide corresponding to SEQ ID NO:10, and the GP100peptide corresponding to SEQ ID NO:11. In some embodiments, the IL-13Rα2peptide-based vaccine is administered concurrently with one or morehelper T cell epitopes. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with a helper T cellepitope, wherein the helper T cell epitope is the PADRE peptide. In someembodiments, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with one or more immune response modifiers. In someembodiments, the IL-13Rα2 peptide-based vaccine is a cell-free vaccine.In other embodiments, the IL-13Rα2 peptide-based vaccine is a dendriticcell vaccine.

In another embodiment, an IL-13Rα2 peptide-based vaccine comprises anIL-13Rα2 peptide, an EphA2 peptide, a survivin peptide, and a WT1peptide. In a specific embodiment, an IL-13Rα2 peptide-based vaccinecomprises the IL-13Rα2 peptide corresponding to any one of SEQ IDNOs:1-4, the EphA2 peptide corresponding to SEQ ID NO:6, the survivinpeptide corresponding to SEQ ID NO:7, and the WT1 peptide correspondingto SEQ ID NO:8. In another specific embodiment, an IL-13Rα2peptide-based vaccine comprises the IL-13Rα2 peptide corresponding toSEQ ID NO:3, the EphA2 peptide corresponding to SEQ ID NO:6, thesurvivin peptide corresponding to SEQ ID NO:7, and the WT1 peptidecorresponding to SEQ ID NO:8. In some embodiments, the IL-13Rα2peptide-based vaccine is administered concurrently with one or morehelper T cell epitopes. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with a helper T cellepitope, wherein the helper T cell epitope is the Tetanus toxoid. Insome embodiments, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with one or more immune response modifiers. In a specificembodiment, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with an immune response modifier, wherein the immuneresponse modifier is poly-ICLC. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with an immuneresponse modifier, wherein the immune response modifier is MontanideISA-51. In some embodiments, the IL-13Rα2 peptide-based vaccine is acell-free vaccine. In other embodiments, the IL-13Rα2 peptide-basedvaccine is a dendritic cell vaccine.

In another embodiment, an IL-13Rα2 peptide-based vaccine comprises anIL-13Rα2 peptide, an EphA2 peptide, and a survivin peptide. In aspecific embodiment, an IL-13Rα2 peptide-based vaccine comprises theIL-13Rα2 peptide corresponding to any one of SEQ ID NOs:1-4, the EphA2peptide corresponding to SEQ ID NO:6, and the survivin peptidecorresponding to SEQ ID NO:7. In another specific embodiment, anIL-13Rα2 peptide-based vaccine comprises the IL-13Rα2 peptidecorresponding to SEQ ID NO:3, the EphA2 peptide corresponding to SEQ IDNO:6, and the survivin peptide corresponding to SEQ ID NO:7. In someembodiments, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with one or more helper T cell epitopes. In a specificembodiment, the IL-13Rα2 peptide-based vaccine is administeredconcurrently with a helper T cell epitope, wherein the helper T cellepitope is the Tetanus toxoid. In some embodiments, the IL-13Rα2peptide-based vaccine is administered concurrently with one or moreimmune response modifiers. In a specific embodiment, the IL-13Rα2peptide-based vaccine is administered concurrently with an immuneresponse modifier, wherein the immune response modifier is poly-ICLC. Ina specific embodiment, the IL-13Rα2 peptide-based vaccine isadministered concurrently with an immune response modifier, wherein theimmune response modifier is Montanide ISA-51. In some embodiments, theIL-13Rα2 peptide-based vaccine is a cell-free vaccine. In otherembodiments, the IL-13Rα2 peptide-based vaccine is a dendritic cellvaccine.

6.1 Peptides

6.1.1 IL-13Rα2 Peptide

IL-13Rα2 a membrane glycoprotein that binds as a component of aheterodimer to the Th2 cytokine, IL-13, which induces monocytes andmacrophages to produce TGFβ (see, e.g., Fichtner-Feigl et al., Nat.Med., 12: 99-106, 2006).

The IL-13Rα2 peptide-based vaccines provided herein comprise an IL-13Rα2peptide. Any IL-13Rα2 peptide capable of serving as an HLA-A2 restrictedcytotoxic T lymphocyte (CTL) epitope may be used in a vaccine describedherein. In some embodiments, the IL-13Rα2 peptide used in a vaccinedescribed herein comprises any one of SEQ ID NOs:1-4. In a specificembodiment, the IL-13Rα2 peptide used in a vaccine described hereincomprises SEQ ID NO:3.

In some embodiments, the IL-13Rα2 peptide used in a vaccine describedherein comprises a mutated version of SEQ ID NO:1, wherein the mutatedversion of SEQ ID NO:1 comprises at least 1, at least 2, or at least 3amino acid substitutions (e.g., conservative substitutions), additions,or deletions.

In some embodiments, the IL-13Rα2 peptide used in a vaccine describedherein comprises an amino acid sequence with at least 50%, 60%, 70%,80%, or 90% identity to SEQ ID NO:1. In other embodiments, the IL-13Rα2peptide used in a vaccine described herein comprises an amino acidsequence with at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%,70% to 90%, or 80% to 90% identity to SEQ ID NO:1. In some embodiments,the IL-13Rα2 peptide used in a vaccine described herein comprises anamino acid sequence with at least 50%, 60%, 70%, 80%, or 90% similarityto SEQ ID NO:1. In other embodiments, the IL-13Rα2 peptide used in avaccine described herein comprises an amino acid sequence with at least50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to90% similarity to SEQ ID NO:1.

6.1.2 EphA2 Peptide

EphA2 is a tyrosine kinase receptor that is involved in the formation ofthe notochord via interaction with ephrinA1. (see, e.g., Naruse-Nakajimaet al., Mech. Dev., 102: 95-105, 2001).

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereincomprise an EphA2 peptide. Any EphA2 peptide capable of serving as anHLA-A2 restricted cytotoxic T lymphocyte (CTL) epitope may be used in avaccine described herein. In some embodiments, the EphA2 peptide used ina vaccine described herein comprises SEQ ID NO:6. In other embodiments,the EphA2 peptide used in a vaccine described herein is an EphA2 peptidedescribed in U.S. Pat. No. 7,297,337.

In some embodiments, the EphA2 peptide used in a vaccine describedherein comprises a mutated version of SEQ ID NO:6, wherein the mutatedversion of SEQ ID NO:6 comprises at least 1, at least 2, or at least 3amino acid substitutions (e.g., conservative substitutions), additions,or deletions.

In some embodiments, the EphA2 peptide used in a vaccine describedherein comprises an amino acid sequence with at least 50%, 60%, 70%,80%, or 90% identity to SEQ ID NO:6. In other embodiments, the EphA2peptide used in a vaccine described herein comprises an amino acidsequence with at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%,70% to 90%, or 80% to 90% identity to SEQ ID NO:6. In some embodiments,the EphA2 peptide used in a vaccine described herein comprises an aminoacid sequence with at least 50%, 60%, 70%, 80%, or 90% similarity to SEQID NO:6. In other embodiments, the EphA2 peptide used in a vaccinedescribed herein comprises an amino acid sequence with at least 50% to60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90%similarity to SEQ ID NO:6.

6.1.3 Survivin Peptide

Survivin is an apoptosis inhibitor protein that is overexpressed in mosthuman cancers, and inhibition of its function results in increasedapoptosis (see, e.g., Blanc-Brude et al., Nat. Med., 8: 987-994, 2002).

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereincomprise a survivin peptide. Any survivin peptide capable of serving asan HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitope may be used ina vaccine described herein. In some embodiments, the survivin peptideused in a vaccine described herein comprises SEQ ID NO:7. In a specificembodiment, the IL-13Rα2 peptide used in a vaccine described hereincomprises SEQ ID NO:7. In other embodiments, the survivin peptide usedin a vaccine described herein is a survivin peptide described in U.S.Application Publication No. 2009/0041732 or by Ciesielski et al., CancerImmunol. Immunother., 59:1211-1221, 2010.

In some embodiments, the survivin peptide used in a vaccine describedherein comprises a mutated version of SEQ ID NO:7, wherein the mutatedversion of SEQ ID NO:7 comprises at least 1, at least 2, or at least 3amino acid substitutions (e.g., conservative substitutions), additions,or deletions.

In some embodiments, the survivin peptide used in a vaccine describedherein comprises an amino acid sequence with at least 50%, 60%, 70%,80%, or 90% identity to SEQ ID NO:7. In other embodiments, the survivinpeptide used in a vaccine described herein comprises an amino acidsequence with at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%,70% to 90%, or 80% to 90% identity to SEQ ID NO:7. In some embodiments,the survivin peptide used in a vaccine described herein comprises anamino acid sequence with at least 50%, 60%, 70%, 80%, or 90% similarityto SEQ ID NO:7. In other embodiments, the survivin peptide used in avaccine described herein comprises an amino acid sequence with at least50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to90% similarity to SEQ ID NO:7.

6.1.4 WT1 Peptide

WT1, is a transcription factor, that is expressed during renaldevelopment and regulates development of the caudal mesonephric tubules(see, e.g., Sainio, Development, 124: 1293-1299, 1997).

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereincomprise a WT1 peptide. Any WT1 peptide capable of serving as an HLA-A2restricted cytotoxic T lymphocyte (CTL) epitope may be used in a vaccinedescribed herein. In some embodiments, the WT1 peptide used in a vaccinedescribed herein comprises SEQ ID NO:8.

In some embodiments, the WT1 peptide used in a vaccine described hereincomprises a mutated version of SEQ ID NO:8, wherein the mutated versionof SEQ ID NO:8 comprises at least 1, at least 2, or at least 3 aminoacid substitutions (e.g., conservative substitutions), additions, ordeletions.

In some embodiments, the WT1 peptide used in a vaccine described hereincomprises an amino acid sequence with at least 50%, 60%, 70%, 80%, or90% identity to SEQ ID NO:8. In other embodiments, the WT1 peptide usedin a vaccine described herein comprises an amino acid sequence with atleast 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80%to 90% identity to SEQ ID NO:8. In some embodiments, the WT1 peptideused in a vaccine described herein comprises an amino acid sequence withat least 50%, 60%, 70%, 80%, or 90% similarity to SEQ ID NO:8. In otherembodiments, the WT1 peptide used in a vaccine described hereincomprises an amino acid sequence with at least 50% to 60%, 50% to 70%,60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90% similarity to SEQ IDNO:8.

6.1.5 GP100 Peptide

Human melanoma-associated antigen, GP100, is a melanocytedifferentiation antigen that is expressed in nucleated mammalian cells.(see, e.g., Koch et al., FEBS Lett., 179: 294-298, 1985.

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereincomprise a GP100 peptide. Any GP100 peptide capable of serving as anHLA-A2 restricted cytotoxic T lymphocyte (CTL) epitope may be used in avaccine described herein. In some embodiments, the GP100 peptide used ina vaccine described herein comprises SEQ ID NO:11.

In some embodiments, the GP100 peptide used in a vaccine describedherein comprises a mutated version of SEQ ID NO:11, wherein the mutatedversion of SEQ ID NO:11 comprises at least 1, at least 2, or at least 3amino acid substitutions (e.g., conservative substitutions), additions,or deletions.

In some embodiments, the GP100 peptide used in a vaccine describedherein comprises an amino acid sequence with at least 50%, 60%, 70%,80%, or 90% identity to SEQ ID NO:11. In other embodiments, the GP100peptide used in a vaccine described herein comprises an amino acidsequence with at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%,70% to 90%, or 80% to 90% identity to SEQ ID NO:11. In some embodiments,the GP100 peptide used in a vaccine described herein comprises an aminoacid sequence with at least 50%, 60%, 70%, 80%, or 90% similarity to SEQID NO:11. In other embodiments, the GP100 peptide used in a vaccinedescribed herein comprises an amino acid sequence with at least 50% to60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90%similarity to SEQ ID NO:11.

6.1.6 YKL-40 Peptide

YKL-40, a secreted glycoprotein, has been known to be involved inextracellular matrix degradation and/or angiogenesis, such as hepaticfibrosis, rheumatoid arthritis and severe osteoarthritis, (see, e.g.,Bigg et al., (2006), J Biol Chem. 281, 21082-95).

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereincomprise a YKL-40 peptide. Any YKL-40 peptide capable of serving as anHLA-A2 restricted cytotoxic T lymphocyte (CTL) epitope may be used in avaccine described herein. In some embodiments, the YKL-40 peptide usedin a vaccine described herein comprises SEQ ID NO:10.

In some embodiments, the YKL-40 peptide used in a vaccine describedherein comprises a mutated version of SEQ ID NO:10, wherein the mutatedversion of SEQ ID NO:10 comprises at least 1, at least 2, or at least 3amino acid substitutions (e.g., conservative substitutions), additions,or deletions.

In some embodiments, the YKL-40 peptide used in a vaccine describedherein comprises an amino acid sequence with at least 50%, 60%, 70%,80%, or 90% identity to SEQ ID NO:10. In other embodiments, the YKL-40peptide used in a vaccine described herein comprises an amino acidsequence with at least 50% to 60%, 50% to 70%, 60% to 70%, 70% to 80%,70% to 90%, or 80% to 90% identity to SEQ ID NO:10. In some embodiments,the YKL-40 peptide used in a vaccine described herein comprises an aminoacid sequence with at least 50%, 60%, 70%, 80%, or 90% similarity to SEQID NO:10. In other embodiments, the YKL-40 peptide used in a vaccinedescribed herein comprises an amino acid sequence with at least 50% to60%, 50% to 70%, 60% to 70%, 70% to 80%, 70% to 90%, or 80% to 90%similarity to SEQ ID NO:10.

6.2 Immune Response Modifiers

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereinare administered concurrently with an immune response modifier. Immuneresponse modifiers include agents capable of modifying the immuneresponse of a subject. In some embodiments, an immune response modifierpolarizes the immune response of a subject toward a Th1 response. Inother embodiments, an immune response modifier polarizes the immuneresponse of a subject toward a Th2 response. In a preferred embodiment,the immune response modified binds to a toll-like receptor, also knownas a TLR, such as TLR3. Exemplary immune response modifiers that can beadministered concurrently with the IL-13Rα2 peptide-based vaccinesprovided herein include, without limitation, poly-ICLC, imiquimod(Aldara®; Beselna®), and MIS-416 (Innate Therapeutics).

6.2.1 Poly-ICLC

Polyinosinic-Polycytidylic acid stabilized with polylysine andcarboxymethylcellulose (poly-ICLC) is a synthetic nucleic acid, andfunctions as a Toll-like receptor-3 (TLR3) ligand. Poly-ICLC is alsoknown as Hiltonol.

6.3 Adjuvants

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereinare administered concurrently with an adjuvant. In some embodiments, theterm “adjuvant” refers to an agent that when administered concurrentlywith or in the same composition as IL-13Rα2 peptide-based vaccinedescribed herein augments, accelerates, prolongs, enhances and/or booststhe immune response to the IL-13Rα2 peptide-based vaccine. In someembodiments, the adjuvant generates an immune response to the IL-13Rα2peptide-based vaccine and does not produce an allergy or other adversereaction. Adjuvants can enhance an immune response by several mechanismsincluding, e.g., lymphocyte recruitment, stimulation of B and/or Tcells, stimulation of dendritic cells and stimulation of macrophages.

Specific examples of adjuvants include, but are not limited to,Montanide ISA-51, Montanide ISA 50V, Montanide, ISA 206, Montanide IMS1312, VaxImmune® (CpG7909; Coley Pharmaceuticals), aluminum salts (alum)(such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate),3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59(Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (seeInternational Application No. PCT/US2007/064857, published asInternational Publication No. WO2007/109812), imidazoquinoxalinecompounds (see International Application No. PCT/US2007/064858,published as International Publication No. WO2007/109813) and saponins,such as QS21 (see Kensil et al., in Vaccine Design: The Subunit andAdjuvant Approach (eds. Powell & Newman, Plenum Press, N Y, 1995); U.S.Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund'sadjuvant (complete or incomplete). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG(Bioworld Today, Nov. 15, 1998). Such adjuvants can be used with orwithout other specific immunostimulating agents such as MPL or 3-DMP,QS21, polymeric or monomeric amino acids such as polyglutamic acid orpolylysine, or other immunopotentiating agents. It should be understoodthat different formulations of IL-13Rα2 peptide-based vaccines maycomprise different adjuvants or may comprise the same adjuvant.

6.4 Helper T Cell Epitopes

In some embodiments, the IL-13Rα2 peptide-based vaccines provided hereinare administered concurrently with a helper T cell epitope. Helper Tcell epitopes include agents that are capable of inducing a helper Tcell response by the immune system. Helper T cells are CD4+ T cells. Insome embodiments, helper T cell epitopes are presented by Class II MHCmolecules, and may be recognized by the T cell receptor (TCR) of helperT cells (CD4+ T cells), thereby activating the CD4+ T cells, causingthem to proliferate, secrete cytokines such as IL2, and activateprofessional antigen presenting cells. Through a variety of mechanisms,activated helper T cells also stimulate killer T cells (also known asCD8+ T cells), thereby prolonging and increasing the CD8+ T cellresponse. Exemplary helper T cell epitopes that can be administeredconcurrently with the IL-13Rα2 peptide-based vaccines provided hereininclude, without limitation, PADRE, HBV core₁₂₈₋₁₄₀, and tetanus toxoid.

6.4.1 PADRE Peptide

PADRE is a non-natural epitope optimized for both HLA-DR binding andT-cell receptor stimulation (see, e.g., Alexander et al, Immunity,1:751-761, 1994).

6.4.2 Tetanus Toxoid

A well characterized Th epitope (SEQ ID NO:9) from the Tetanus Toxoid(TT) protein, to which the vast majority of the population has beensensitized, is known to act as a helper T cell epitope.

6.4.2.1 HBV Core₁₂₈₋₁₄₀

A well characterized Th epitope (SEQ ID NO:5) from the HBV protein isknown to act as a helper T cell epitope.

6.5 Production and Purification of Peptides

The peptides described herein can be produced by any method known in theart for the synthesis of peptides, in particular, by chemical synthesisor by recombinant expression techniques. The methods provided hereinencompass, unless otherwise indicated, conventional techniques inmolecular biology, microbiology, genetic analysis, recombinant DNA,organic chemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described in the references citedherein and are fully explained in the literature. See, e.g., Maniatis etal. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press; Sambrook et al. (1989), Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press;Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren et al. (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

6.5.1.1 Synthetic Production of Peptides

The peptides described herein may be prepared using conventionalstep-wise solution or solid phase synthesis (see, e.g., ChemicalApproaches to the Synthesis of Peptides and Proteins, Williams et al.,Eds., 1997, CRC Press, Boca Raton Fla., and references cited therein;Solid Phase Peptide Synthesis: A Practical Approach, Atherton &Sheppard, Eds., 1989, IRL Press, Oxford, England, and references citedtherein).

Alternatively, the peptides described herein may be prepared by way ofsegment condensation, as described, for example, in Liu et al., 1996,Tetrahedron Lett. 37(7):933-936; Baca, et al., 1995, J. Am. Chem. Soc.117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and Tam,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334. Other methods useful for synthesizingthe peptides described herein are described in Nakagawa et al., 1985, J.Am. Chem. Soc. 107:7087-7092.

Formation of disulfide linkages, if desired, is generally conducted inthe presence of mild oxidizing agents. Chemical oxidizing agents may beused, or the compounds may simply be exposed to atmospheric oxygen toeffect these linkages. Various methods are known in the art, includingthose described, for example, by Tam et al., 1979, Synthesis 955-957;Stewart et al., 1984, Solid Phase Peptide Synthesis, 2d Ed., PierceChemical Company Rockford, Ill.; Ahmed et al., 1975, J. Biol. Chem.250:8477-8482; and Pennington et al., 1991 Peptides 1990 164-166, Giraltand Andreu, Eds., ESCOM Leiden, The Netherlands. An additionalalternative is described by Kamber et al., 1980, Helv. Chim. Acta63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97, each of which isincorporated by reference herein in its entirety.

6.5.1.2 Recombinant Expression of Peptides

Recombinant expression of a peptide requires construction of anexpression vector containing a polynucleotide that encodes the peptide.Once a polynucleotide encoding a peptide has been obtained, the vectorfor the production of the peptide may be produced by recombinant DNAtechnology using techniques well-known in the art. Thus, methods forpreparing a peptide by expressing a polynucleotide containing apeptide-encoding nucleotide sequence are described herein. Methods whichare well known to those skilled in the art can be used to constructexpression vectors containing peptide coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Thus, provided herein arereplicable expression vectors comprising a nucleotide sequence encodinga peptide operably linked to a promoter.

An expression vector comprises a nucleic acid encoding a peptide in aform suitable for expression of the nucleic acid in a host cell. Inspecific embodiments, the host cell is an isolated host cell. In aspecific embodiment, an expression vector includes one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operably linked to the nucleic acid to beexpressed. Within an expression vector, “operably linked” is intended tomean that a nucleic acid of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleic acid(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). Regulatory sequencesinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Regulatory sequences include thosewhich direct constitutive expression of a nucleic acid in many types ofhost cells, those which direct expression of the nucleic acid only incertain host cells (e.g., tissue-specific regulatory sequences), andthose which direct the expression of the nucleic acid upon stimulationwith a particular agent (e.g., inducible regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The term “host cell” is intended to include a particular subject celltransformed or transfected with a nucleic acid and the progeny orpotential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transformed or transfected with the nucleicacid due to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid into the hostcell genome. In specific embodiments, the host cell is isolated.

An expression vector can be introduced into host cells via conventionaltransformation or transfection techniques. Such techniques include, butare not limited to, calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, andelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, andother laboratory manuals. In certain embodiments, a host cell istransiently transfected with an expression vector containing a nucleicacid encoding a peptide. In other embodiments, a host cell is stablytransfected with an expression vector containing a nucleic acid encodinga peptide. Thus, provided herein are host cells containing apolynucleotide encoding a peptide described herein or generated inaccordance with the methods provided herein.

A variety of host-expression vector systems may be utilized to express apeptide. Such host-expression systems represent vehicles by which thecoding sequences of interest may be produced and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, express a peptide in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining peptide coding sequences; yeast (e.g., Saccharomyces Pichia)transformed with recombinant yeast expression vectors containing peptidecoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing peptide codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing peptide coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells are used for the expressionof a peptide. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for peptides (Foecking et al., 1986, Gene 45:101; andCockett et al., 1990, Bio/Technology 8:2). In a specific embodiment, theexpression of nucleotide sequences encoding the peptides describedherein or generated in accordance with the methods provided herein isregulated by a constitutive promoter, inducible promoter or tissuespecific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the peptidebeing expressed. For example, when a large quantity of peptide is to beproduced, for the generation of pharmaceutical compositions of apeptide, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to, the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO 12:1791), in which the peptide codingsequence may be ligated individually into the vector in frame with thelac Z coding region so that a fusion protein is produced; pIN vectors(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke &Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectorsmay also be used to express foreign polypeptides as fusion proteins withglutathione 5-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to matrix glutathione agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The peptide coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the peptide coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the peptide in infected hosts (e.g., see Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals mayalso be required for efficient translation of inserted peptide codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the peptide. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, Vero,MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant peptides, stableexpression is preferred. For example, cell lines which stably expressthe peptide molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the peptide.Such engineered cell lines may be particularly useful in screening andevaluation of compositions that interact directly or indirectly with thepeptide. Methods commonly known in the art of recombinant DNA technologymay be routinely applied to select the desired recombinant clone, andsuch methods are described, for example, in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993);Kriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, N Y (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.),Current Protocols in Human Genetics, John Wiley & Sons, N Y (1994);Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which areincorporated by reference herein in their entireties.

The expression levels of a peptide can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York,1987)). When a marker in the vector system expressing the peptide isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the peptide, production of thepeptide will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

As an alternative to recombinant expression of a peptide using a hostcell, an expression vector containing a nucleic acid encoding a peptidecan be transcribed and translated in vitro using, e.g., T7 promoterregulatory sequences and T7 polymerase. In a specific embodiment, acoupled transcription/translation system, such as Promega TNT®, or acell lysate or cell extract comprising the components necessary fortranscription and translation may be used to produce a peptide.

Accordingly, provided herein are methods for producing a peptide. In oneembodiment, the method comprises culturing a host cell containing anucleic acid encoding the peptide in a suitable medium such that thepeptide is produced. In some embodiments, the method further comprisesisolating the peptide from the medium or the host cell.

In certain embodiments, plants (e.g., plants of the genus Nicotiana) maybe engineered to express a peptide described herein. In specificembodiments, plants are engineered to express a peptide described hereinvia an agroinfiltration procedure using methods known in the art. Forexample, nucleic acids encoding a gene of interest, e.g., a geneencoding a peptide described herein, are introduced into a strain ofAgrobacterium. Subsequently the strain is grown in a liquid culture andthe resulting bacteria are washed and suspended into a buffer solution.The plants are then exposed (e.g., via injection or submersion) to theAgrobacterium that comprises the nucleic acids encoding a peptidedescribed herein such that the Agrobacterium transforms the gene ofinterest to a portion of the plant cells. The peptide is thentransiently expressed by the plant and can be isolated using methodsknown in the art and described herein. (For specific examples see Shojiet al., 2008, Vaccine, 26(23):2930-2934; and D'Aoust et al., 2008, J.Plant Biotechnology, 6(9):930-940). In a specific embodiment, the plantis a tobacco plant (i.e., Nicotiana tabacum). In another specificembodiment, the plant is a relative of the tobacco plant (e.g.,Nicotiana benthamiana).

In other embodiments, algae (e.g., Chlamydomonas reinhardtii) may beengineered to express a peptide described herein (see, e.g., Rasala etal., 2010, Plant Biotechnology Journal (Published online Mar. 7, 2010)).

6.5.1.3 Purification of Peptides

The peptides described herein and generated using the approachesdescribed in may be purified by any method known in the art forpurification of a peptide, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. Further, the peptides may be fused toheterologous peptide sequences described herein or otherwise known inthe art to facilitate purification. The actual conditions used to purifya particular peptide will depend, in part, on the synthesis strategy(e.g., synthetic production vs. recombinant production) and on factorssuch as net charge, hydrophobicity, and/or hydrophilicity of thepeptide, and will be apparent to those having skill in the art.

6.6 Pharmaceutical Compositions and Routes of Administration

Provided herein are pharmaceutical compositions comprising. In someembodiments, a composition provided herein comprises an interleukin-13receptor α2 peptide-based brain cancer vaccine. In other embodiments, acomposition provided herein comprises an IL-13Rα2 peptide-based vaccineand a helper T cell epitope, an adjuvant, and/or an immune responsemodifier. In other embodiments, a composition provided herein comprisesan immune response modifier. The pharmaceutical compositions providedherein are suitable for veterinary and/or human administration.

The pharmaceutical compositions provided herein (e.g., a compositioncomprising an IL-13Rα2 peptide-based vaccine, a composition comprisingan IL-13Rα2 peptide-based vaccine and a helper T cell epitope, anadjuvant, and/or an immune response modifier, or a compositioncomprising an immune response modifier) can be in any form that allowsfor the composition to be administered to a subject, said subjectpreferably being an animal, including, but not limited to a human,mammal, or non-human animal, such as a cow, horse, sheep, pig, fowl,cat, dog, mouse, rat, rabbit, guinea pig, etc., and is more preferably amammal, and most preferably a human.

In specific embodiments, the compositions provided herein (e.g., acomposition comprising an IL-13Rα2 peptide-based vaccine, a compositioncomprising an IL-13Rα2 peptide-based vaccine and a helper T cellepitope, an adjuvant, and/or an immune response modifier, or acomposition comprising an immune response modifier) are in the form of aliquid (e.g., an elixir, syrup, solution, emulsion, or suspension).Typical routes of administration of the liquid compositions providedherein may include, without limitation, parenteral, intradermal,intratumoral, intracerebral, and intrathecal. Parenteral administrationincludes, without limitation, subcutaneous, intranodal, intravenous,intramuscular, intraperitoneal, and intrapleural administrationtechniques. In a specific embodiment, the compositions are administeredparenterally. In a composition for administration by injection, one ormore of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer, and isotonic agent may beincluded. In a specific embodiment, a pump may be used to deliver thevaccines (see, e.g, Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14, 201;Buchwald et al., Surgery 1980, 88: 507; Saudek et al., N. Engl. J. Med.1989, 321: 574). In a specific embodiment, the pump may be, but is notlimited to, an insulin-like pump.

Materials used in preparing the pharmaceutical compositions providedherein (e.g., a composition comprising an IL-13Rα2 peptide-basedvaccine, a composition comprising an IL-13Rα2 peptide-based vaccine anda helper T cell epitope, an adjuvant, and/or an immune responsemodifier, or a composition comprising an immune response modifier) canbe non-toxic in the amounts used. It may be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of subject (e.g.,human), the overall health of the subject, the type of brain cancer thesubject is in need of treatment of, the use of the composition as partof a multi-drug regimen, the particular form of the vaccine beingadministered, the manner of administration, and the compositionemployed.

The liquid compositions of the invention, whether they are solutions,suspensions, or other like form, can also include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or digylcerides whichcan serve as the solvent or suspending medium, polyethylene glycols,glycerin, cyclodextrin, propylene glycol, or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates, or phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin an ampoule, a disposable syringe, or a multiple-dose vial made ofglass, plastic or other material. An injectable composition ispreferably sterile.

The compositions provided herein (e.g., a composition comprising anIL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2peptide-based vaccine and a helper T cell epitope, an adjuvant, and/oran immune response modifier, or a composition comprising an immuneresponse modifier) may comprise a pharmaceutically acceptable carrier orvehicle. As used herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeiae for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the pharmaceutical composition is administered. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Suitableexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. Examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. The formulation should suit the mode of administration.

In one embodiment, the compositions provided herein (e.g., a compositioncomprising an IL-13Rα2 peptide-based vaccine, a composition comprisingan IL-13Rα2 peptide-based vaccine and a helper T cell epitope, anadjuvant, and/or an immune response modifier, or a compositioncomprising an immune response modifier) are formulated in accordancewith routine procedures as a pharmaceutical composition adapted forparenteral administration to animals, particularly human beings.Generally, the ingredients in the compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachet indicating the quantity of activeagent. Where a composition described herein is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration,if necessary.

The compositions provided herein (e.g., a composition comprising anIL-13Rα2 peptide-based vaccine, a composition comprising an IL-13Rα2peptide-based vaccine and a helper T cell epitope, an adjuvant, and/oran immune response modifier, or a composition comprising an immuneresponse modifier) described herein can comprise an additional activeagent selected from among those including, but not limited to, anadditional prophylactic agent, an additional therapeutic agent, anantiemetic agent, a hematopoietic colony stimulating factor, an adjuvanttherapy, an antibody/antibody fragment-based agent, an anti-depressantand an analgesic agent.

The pharmaceutical compositions provided herein (e.g., a compositioncomprising an IL-13Rα2 peptide-based vaccine, a composition comprisingan IL-13Rα2 peptide-based vaccine and a helper T cell epitope, anadjuvant, and/or an immune response modifier, or a compositioncomprising an immune response modifier) can be prepared usingmethodology well known in the pharmaceutical art. For example, acomposition intended to be administered by injection can be prepared bycombining the peptides of a vaccine described herein with water and/orother liquid components so as to form a solution. A surfactant can beadded to facilitate the formation of a homogeneous solution orsuspension.

The pharmaceutical compositions described herein can be included in acontainer, pack, or dispenser together with instructions foradministration.

6.7 Prophylactic and Therapeutic Uses

In one aspect, provided herein are methods for preventing, treating,and/or managing brain cancer in a subject in need thereof byadministering an effective amount of an IL-13Rα2 peptide-based vaccinedescribed herein.

In another aspect, provided herein is a method of preventing, treating,and/or managing brain cancer in a patient (e.g., a human patient), themethod comprising administering to the patient a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient an IL-13Rα2 peptide-basedvaccine described herein or a pharmaceutical composition describedherein, wherein the patient has been diagnosed with brain cancer.

In another aspect, provided herein is a method of preventing, treating,and/or managing brain cancer in a patient (e.g., a human patient), themethod comprising administering to the patient a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient an IL-13Rα2 peptide-basedvaccine described herein or a pharmaceutical composition describedherein, wherein the patient has relapsed from brain cancer.

In another aspect, provided herein is a method of preventing, treating,and/or managing brain cancer in a patient (e.g., a human patient), themethod comprising administering to the patient a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient an IL-13Rα2 peptide-basedvaccine described herein or a pharmaceutical composition describedherein, wherein the patient has failed or is failing brain cancertherapy that does not comprise a vaccine described herein.

In another aspect, provided herein is a method of preventing, treating,and/or managing brain cancer in a patient (e.g., a human patient), themethod comprising administering to the patient a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient an IL-13Rα2 peptide-basedvaccine described herein or a pharmaceutical composition describedherein, wherein the patient is in remission from brain cancer.

In another aspect, provided herein is a method of preventing, treating,and/or managing brain cancer in a patient (e.g., a human patient), themethod comprising administering to the patient a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient an IL-13Rα2 peptide-basedvaccine described herein or a pharmaceutical composition describedherein, wherein the patient is refractory to brain cancer therapy thatdoes not comprise a vaccine described herein. In one embodiment of thisaspect, the patient has received or is receiving brain cancer therapythat does not comprise a vaccine described herein. In another embodimentof this aspect, the patient has not previously received a brain cancertherapy that does not comprise a vaccine described herein for theprevention, treatment, and/or management of the brain cancer.

In another aspect, provided herein is a method of preventing, treating,and/or managing brain cancer in a patient (e.g., a human patient), themethod comprising administering to the patient a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient an IL-13Rα2 peptide-basedvaccine described herein or a pharmaceutical composition describedherein, wherein the patient has received another brain cancer therapy.In some embodiments, the prior brain cancer therapy is, for example,chemotherapy, radiation therapy, surgical therapy, small moleculetherapy, biologic therapy, antibody therapy, hormone therapy,immunotherapy, anti-angiogenic therapy or any combination thereof. Insome embodiments, the prior therapy has failed in the patient. In someembodiments, the therapeutically effective regimen comprisingadministration of an IL-13Rα2 peptide-based vaccine described herein isadministered to the patient immediately after the patient has undergonethe prior therapy. For instance, in certain embodiments, the outcome ofthe prior therapy may be unknown before the patient is administered theIL-13Rα2 peptide-based vaccine. In one embodiment, the priorchemotherapy is temolozimide. In embodiment, the prior therapy isradiation therapy. In another embodiment, the prior therapy is acombination of temozolomide and radiation therapy. In a preferredembodiment, the combination of temozolomide and radiation areadministered using the Stupp regimen. In another embodiment, the priortherapy is surgery. In some embodiments, the patient undergoes surgerybefore the initiation of combination therapy. In some embodiments, thepatient undergoes surgery before treatment with temozolomide. In someembodiments, the patient undergoes surgery before the initiation ofradiation therapy. In each of these embodiments that describe the use ofcombination therapy, the IL-13Rα2 peptide-based vaccine may beadministered before, during, or after the treatment of the patient withthe therapy that is being combined.

In some embodiments, the IL-13Rα2 peptide-based vaccines describedherein are administered as monotherapy for the prevention, treatment,and/or management of brain cancer. In other embodiments, provided hereinare methods comprising administering to a subject in need thereof anIL-13Rα2 peptide-based vaccine described herein and one or more agentsother than the IL-13Rα2 peptide-based vaccine described herein that arecurrently being used, have been used, are known to be useful, or may beuseful in the prevention, treatment, and/or management of brain canceror one or more symptoms thereof. The agents of the combination therapiescan be administered sequentially or concurrently. In certainembodiments, the combination therapies improve the prophylactic ortherapeutic effect of an IL-13Rα2 peptide-based vaccine described hereinfunctioning together with the IL-13Rα2 peptide-based vaccine describedherein to have an additive or synergistic effect. In some embodiments,the combination therapies are administered prior to, during, or afterthe administration of the compositions described herein.

In another aspect, provided herein are methods for inducing an immuneresponse in a subject with brain cancer comprising administering aneffective amount of an IL-13Rα2 peptide-based vaccine described herein.In some embodiments, the immune response induced in a subject by anIL-13Rα2 peptide-based vaccine described herein or a compositiondescribed herein is effective to prevent, treat, and/or manage braincancer in the subject. In some embodiments, the immune response inducedin a subject by an IL-13Rα2 peptide-based vaccine described herein or acomposition described herein is effective to reduce symptoms of braincancer in the subject.

The medical practitioner can diagnose the patient using any of theconventional brain cancer screening methods including, but not limitedto neurological examination; imaging methods (e.g., computed tomography(C_(T)), magnetic resonance imaging (MRI), ultrasound, X-ray imaging,and positron emission tomography (PET) scans); and biopsy (e.g.,sterotactic biopsy).

6.7.1 Dosage and Frequency of Administration

The amount of a composition described herein (e.g., a compositioncomprising an IL-13Rα2 peptide-based vaccine, a composition comprisingan IL-13Rα2 peptide-based vaccine and a helper T cell epitope, anadjuvant, and/or an immune response modifier, or a compositioncomprising an immune response modifier) which will be effective in thetreatment, prevention, and or management of brain cancer may depend onthe status of the brain cancer, the patient to whom the composition(s)is to be administered, the route of administration, and/or the type ofbrain cancer. Such doses can be determined by standard clinicaltechniques and may be decided according to the judgment of thepractitioner.

For example, effective doses may vary depending upon means ofadministration, target site, physiological state of the patient(including age, body weight, health), whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages are optimally titrated to optimize safety andefficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

In certain embodiments, the IL-13Rα2 peptide-based vaccine is acell-free vaccine, wherein the cell-free vaccine comprises an IL-13Rα2peptide and one, two, three, or more additional brain cancer-associatedpeptides. In some embodiments, exemplary cell-free IL-13Rα2peptide-based vaccines comprise about 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550,600, 650, 700, 750, or 800 μg of each brain cancer-associated peptideper dose. In other embodiments, exemplary cell-free IL-13Rα2peptide-based vaccines comprise about 25 to 50, 25 to 75, 25 to 100, 50to 100, 50 to 150, 50 to 200, 100 to 150, 100 to 200, 100 to 250, 100 to300, 150 to 200, 150 to 250, 150 to 300, 200 to 250, 250 to 300, 250 to350, 250 to 400, 300 to 350, 300 to 400, 300 to 450, 300 to 500, 350 to400, 350 to 450, 400 to 500, 400 to 600, 500 to 600, 500 to 700, 600 to700, 600 to 800, or 700 to 800 μg of each brain cancer-associatedpeptide per dose. In other embodiments, exemplary cell-free IL-13Rα2peptide-based vaccines comprise about 5 μg to 100 mg, 15 μg to 50 mg, 15μg to 25 mg, 15 μg to 10 mg, 15 μg to 5 mg, 15 μg to 1 mg, 15 μg to 100μg, 15 μg to 75 μg, 5 μg to 50 μg, 10 μg to 50 μg, 15 μg to 45 μg, 20 μgto 40 μg, or 25 to 35 μg of each brain cancer-associated peptide perkilogram of the patient.

In certain embodiments, the cell-free IL-13Rα2 peptide-based vaccinesare administered concurrently with a helper T cell epitope. In someembodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines areadministered concurrently with about 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, or600 μg of a helper T cell epitope. In other embodiments, exemplarycell-free IL-13Rα2 peptide-based vaccines are administered concurrentlywith about 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 to 150, 50 to200, 100 to 150, 100 to 200, 100 to 250, 100 to 300, 150 to 200, 150 to250, 150 to 300, 200 to 250, 250 to 300, 250 to 350, 250 to 400, 300 to350, 300 to 400, 300 to 450, 300 to 500, 350 to 400, 350 to 450, 400 to500, 400 to 600, or 500 to 600 μg of a helper T cell epitope.

In certain embodiments, the cell-free IL-13Rα2 peptide-based vaccinesare administered concurrently with an immune response modifier. In someembodiments, exemplary cell-free IL-13Rα2 peptide-based vaccines areadministered concurrently with about 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 μg ofan immune response modifier. In other embodiments, exemplary cell-freeIL-13Rα2 peptide-based vaccines are administered concurrently with about100 to 300, 200 to 400, 400 to 800, 600 to 800, 800 to 1000, 800 to1200, 1000 to 1200, 1000 to 1400, 1200 to 1400, 1200 to 1600, 1400 to1600, 1400 to 1800, or 1600 to 1800 μg of an immune response modifier.In other embodiments, exemplary cell-free IL-13Rα2 peptide-basedvaccines are administered concurrently with about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, of 60 μg of an immune response modifer perkilogram of the patient. In other embodiments, exemplary cell-freeIL-13Rα2 peptide-based vaccines are administered concurrently with about1 to 5, 1 to 10, 5 to 10, 5 to 15, 10 to 15, 10 to 20, 15 to 20, 15 to25, 15 to 30, 20 to 25, 20 to 30, 20 to 35, 25 to 30, 25 to 35, 25 to40, 30 to 35, 30 to 40, 35 to 40, 35 to 45, 40 to 45, 40 to 50, 45 to50, 50 to 55, or 50 to 60 μg of an immune response modifier per kilogramof the patient.

In certain embodiments, the cell-free IL-13Rα2 peptide-based vaccinesare administered concurrently with an adjuvant. In some embodiments, acomposition comprising a cell-free IL-13Rα2 peptide-based vaccine ismixed 0.5 to 1, 1 to 0.5, 1 to 1, 1 to 2, 1 to 3, 2 to 1, or 3 to 1 withan adjuvant.

In certain embodiments, the IL-13Rα2 peptide-based vaccine is adendritic cell-based vaccine, wherein the dendritic cell-based vaccinecomprises dendritic cells loaded with an IL-13Rα2 peptide and dendriticcells loaded with one, two, three, or more additional braincancer-associated peptides. In some embodiments, exemplary dendriticcell-based IL-13Rα2 peptide-based vaccines comprise about 10³, 5×10³,10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 3×10⁷, 5×10⁷, 7×10⁷, 10⁸,5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² dendriticcells loaded with brain cancer-associated peptide(s) per dose. In otherembodiments, exemplary dendritic cell-based IL-13Rα2 peptide-basedvaccines comprise about 10³ to 10⁴, 10³ to 10⁵, 10⁴ to 10⁵, 10⁴ to 10⁶,10⁵ to 10⁶, 10⁵ to 10⁷, 10⁶ to 10⁷, 10⁶ to 10⁸, 10⁷ to 10⁸, 10⁷ to 10⁹,10⁸ to 10⁹, 10⁹ to 10¹⁰, 10¹⁰ to 10¹¹, or 10¹¹ to 10¹² dendritic cellsloaded with brain cancer-associated peptide(s) per dose.

In certain embodiments, the dendritic cell-based IL-13Rα2 peptide-basedvaccines are administered concurrently with a helper T cell epitope. Insome embodiments, exemplary dendritic cell-based IL-13Rα2 peptide-basedvaccines are administered concurrently with about 25, 50, 75, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500, 550, or 600 μg of a helper T cell epitope. In other embodiments,exemplary dendritic cell-based IL-13Rα2 peptide-based vaccines areadministered concurrently with about 25 to 50, 25 to 75, 25 to 100, 50to 100, 50 to 150, 50 to 200, 100 to 150, 100 to 200, 100 to 250, 100 to300, 150 to 200, 150 to 250, 150 to 300, 200 to 250, 250 to 300, 250 to350, 250 to 400, 300 to 350, 300 to 400, 300 to 450, 300 to 500, 350 to400, 350 to 450, 400 to 500, 400 to 600, or 500 to 600 μg of a helper Tcell epitope.

In certain embodiments, the dendritic cell-based IL-13Rα2 peptide-basedvaccines are administered concurrently with an immune response modifier.In some embodiments, exemplary dendritic cell-based IL-13Rα2peptide-based vaccines are administered concurrently with about 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, or 1800 μg of an immune response modifier. In otherembodiments, exemplary dendritic cell-based IL-13Rα2 peptide-basedvaccines are administered concurrently with about 100 to 300, 200 to400, 400 to 800, 600 to 800, 800 to 1000, 800 to 1200, 1000 to 1200,1000 to 1400, 1200 to 1400, 1200 to 1600, 1400 to 1600, 1400 to 1800, or1600 to 1800 μg of an immune response modifier. In other embodiments,exemplary dendritic cell-based IL-13Rα2 peptide-based vaccines areadministered concurrently with about 1, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, of 60 μg of an immune response modifer per kilogram of thepatient. In other embodiments, exemplary dendritic cell-based IL-13Rα2peptide-based vaccines are administered concurrently with about 1 to 5,1 to 10, 5 to 10, 5 to 15, 10 to 15, 10 to 20, 15 to 20, 15 to 25, 15 to30, 20 to 25, 20 to 30, 20 to 35, 25 to 30, 25 to 35, 25 to 40, 30 to35, 30 to 40, 35 to 40, 35 to 45, 40 to 45, 40 to 50, 45 to 50, 50 to55, or 50 to 60 μg of an immune response modifier per kilogram of thepatient.

In certain embodiments, the dendritic cell-based IL-13Rα2 peptide-basedvaccines are administered concurrently with an adjuvant. In someembodiments, a composition comprising a dendritic cell-based IL-13Rα2peptide-based vaccine is mixed 0.5 to 1, 1 to 0.5, 1 to 1, 1 to 2, 1 to3, 2 to 1, or 3 to 1 with an adjuvant.

In certain embodiments, a composition described herein (e.g., acomposition comprising an IL-13Rα2 peptide-based vaccine, a compositioncomprising an IL-13Rα2 peptide-based vaccine and a helper T cellepitope, an adjuvant, and/or an immune response modifier, or acomposition comprising an immune response modifier) is administered to asubject once as a single dose. In some embodiments, a compositiondescribed herein (e.g., a composition comprising an IL-13Rα2peptide-based vaccine, a composition comprising an IL-13Rα2peptide-based vaccine and a helper T cell epitope, an adjuvant, and/oran immune response modifier, or a composition comprising an immuneresponse modifier) is administered in multiple doses (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 doses), wherein the doses may beseparated by at least 1 day, 2 days, 3 days, 4, days 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 15 days, or 30 days. In specificembodiments, the IL-13Rα2 peptide-based vaccine is administeredintranodally or subcutaneously and the immune response modifier isadministered intramuscularly.

In some embodiments, when a composition described herein comprises acell-free IL-13Rα2 peptide-based vaccine, the composition may beadministered over the course of 21 weeks, with administrations occurringon weeks 0, 3, 6, 9, 12, 15, 18 and 21. In certain embodiments, thecomposition comprising a cell-free IL-13Rα2 peptide-based vaccine isadministered concurrently with a helper T cell epitope, an adjuvant,and/or an immune response modifer. In a specific embodiment, acomposition described herein comprising a cell-free IL-13Rα2peptide-based vaccine is administered over the course of 21 weeks, withadministrations occurring on weeks 0, 3, 6, 9, 12, 15, 18 and 21, andthe composition is administered concurrently with an immune responsemodifier, wherein the immune response modifier is administered on theday of each administration of the cell-free IL-13Rα2 peptide-basedvaccine and on day 4 after each administration of the cell-free IL-13Rα2peptide-based vaccine. In another specific embodiment, a compositiondescribed herein comprising a cell-free IL-13Rα2 peptide-based vaccineis administered over the course of 21 weeks, with administrationsoccurring on weeks 0, 3, 6, 9, 12, 15, 18 and 21, and the composition isadministered concurrently with an immune response modifier, wherein theimmune response modifier is administered on the day of eachadministration of the cell-free IL-13Rα2 peptide-based vaccine. Inspecific embodiments, the cell-free IL-13Rα2 peptide-based vaccine isadministered subcutaneously and the immune response modifier isadministered intramuscularly.

In some embodiments, when a composition described herein comprises adendritic cell-based IL-13Rα2 peptide-based vaccine, the composition maybe administered over the course of 6 weeks, with administrationsoccurring on weeks 0, 2, 4, and 6. In certain embodiments, thecomposition comprising a cell-free IL-13Rα2 peptide-based vaccine isadministered concurrently with a helper T cell epitope, an adjuvant,and/or an immune response modifer. In a specific embodiment, acomposition described herein comprising a dendritic cell-based IL-13Rα2peptide-based vaccine is administered over the course of 6 weeks, withadministrations occurring on weeks 0, 2, 4, and 6, and the compositionis administered concurrently with an immune response modifier, whereinthe immune response modifier is administered twice per week beginning onthe first day of administration of the dendritic cell-based IL-13Rα2peptide-based vaccine. In specific embodiments, the dendritic cell-basedIL-13Rα2 peptide-based vaccine is administered intranodally and theimmune response modifier is administered intramuscularly.

In some embodiments, when a composition described herein comprises adendritic cell-based IL-13Rα2 peptide-based vaccine, the composition maybe administered over the course of 26 weeks, with administrationsoccurring on weeks 0, 2, 4, 6, 10, 14, 18, 22, and 26. In certainembodiments, the composition comprising a cell-free IL-13Rα2peptide-based vaccine is administered concurrently with a helper T cellepitope, an adjuvant, and/or an immune response modifer. In a specificembodiment, a composition described herein comprising a dendriticcell-based IL-13Rα2 peptide-based vaccine is administered over thecourse of 26 weeks, with administrations occurring on weeks 0, 2, 4, 6,10, 14, 18, 22, and 26, and the composition is administered concurrentlywith an immune response modifier, wherein the immune response modifieris administered twice per week beginning on the first day ofadministration of the dendritic cell-based IL-13Rα2 peptide-basedvaccine. In specific embodiments, the dendritic cell-based IL-13Rα2peptide-based vaccine is administered intranodally and the immuneresponse modifier is administered intramuscularly.

6.7.2 Brain Cancers

The IL-13Rα2 peptide-based vaccine described herein can be used in theprevention, treatment, and/or management of brain cancer. Any type ofbrain cancer can be treated with the IL-13Rα2 peptide-based vaccinesdescribed herein in accordance with the methods described herein.Exemplary brain cancers include, but are not limited to, gliomas(including astrocytoma (e.g., pilocytic astrocytoma, diffuseastrocytoma, and anaplastic astrocytoma), glioblastoma,oligodendroglioma, brain stem glioma, non-brain stem glioma, ependymoma,and mixed tumors comprising more than one glial cell types), acousticschwannoma, cranialpharyngioma, meningioma, medulloblastoma, primarycentral nervous system lymphoma, and tumors of the pineal (e.g., pinealastrocytic tumors and pineal parenchymal tumors) and pituitary glands.Gliomas additionally include recurrent malignant gliomas, high-risk WHOGrade II Astrocytomas, Oligo Astrocytomas, recurrent WHO Grade IIGliomas, newly-diagnosed malignant or intrinsic brain stem gliomas,incompletely resected non-brainstem gliomas, and recurrent unresectablelow-grade gliomas. Additional types of brain cancer that can be treatedwith the IL-13Rα2 peptide-based vaccines described herein in accordancewith the methods described herein include adult low-grade infiltrativesupratentorial astrocytoma/oligodendroglioma, adult low-gradeinfiltrative supratentorial astrocytoma, adult low-grade infiltrativesupratentorial oligodendroglioma, adult low-grade infiltrativesupratentorial astrocytoma/oligodendroglioma (excluding pilocyticastrocytoma), adult low-grade infiltrative supratentorial astrocytoma(excluding pilocytic astrocytoma), adult low-grade infiltrativesupratentorial oligodendroglioma (excluding pilocytic astrocytoma),adult intracranial ependymoma, adult intracranial ependymoma (excludingsubependymoma and myxopapillary), adult intracranial anaplasticependymoma, anaplastic glioma, anaplastic glioblastoma, pilocyticastrocytoma, subependymoma, myxopapillary, 1 to 3 limited metastaticlesions (intraparenchymal), greater than 3 metastatic lesions(intraparenchymal), leptomeningeal metastases (neoplastic meningitis),primary CNS lymphoma, metastatic spine tumors, or meningiomas.

In one embodiment, the brain cancer treated with the IL-13Rα2peptide-based vaccines described herein in accordance with the methodsdescribed herein is a glioma. In a specific embodiment, the brain cancertreated with the IL-13Rα2 peptide-based vaccines described herein inaccordance with the methods described herein is recurrent malignantglioma. In another specific embodiment, the brain cancer treated withthe IL-13Rα2 peptide-based vaccines described herein in accordance withthe methods described herein is recurrent WHO Grade II Glioma. Inanother specific embodiment, the brain cancer treated with the IL-13Rα2peptide-based vaccines described herein in accordance with the methodsdescribed herein is newly-diagnosed malignant or intrinsic brain stemglioma. In another specific embodiment, the brain cancer treated withthe IL-13Rα2 peptide-based vaccines described herein in accordance withthe methods described herein is incompletely resected non-brainstemglioma. In another specific embodiment, the brain cancer treated withthe IL-13Rα2 peptide-based vaccines described herein in accordance withthe methods described herein is recurrent unresectable low-grade glioma.In one embodiment, the patient is an adult with recurrent malignantglioma, recurrent glioblastoma, anaplastic astrocytoma, anaplasticoligodendroglioma, or anaplastic mixed oligoastrocytoma. In anotherspecific embodiment, the patient is an adult with newly diagnosedhigh-risk low grade glioma. In another specific embodiment, the patientis an adult with newly diagnosed high-risk low grade astrocytoma. Inanother specific embodiment, the patient is an adult with newlydiagnosed high-risk low grade oligoastrocytoma. In another specificembodiment, the patient is an adult with recurrent high-risk low gradeastrocytoma. In another specific embodiment, the patient is an adultwith recurrent high-risk low grade oligoastrocytoma. In another specificembodiment, the patient is an adult with recurrent high-risk low gradeoligodendroglioma. In another specific embodiment, the patient is achild with newly diagnosed malignant glioma. In another specificembodiment, the patient is a child with intrinsic brain stem glioma. Inanother specific embodiment, the patient is a child with incompletelyresected non-brainsteam high-grade glioma. In another specificembodiment, the patient is a child with recurrent unresectable low-gradeglioma. In another specific embodiment, the patient is a child withnewly diagnosed diffuse intrinsic pontine glioma. In another specificembodiment, the patient is a child with any high-grade glioma involvingthe brainstem and treated with RT or without chemotherapy during RT. Inanother specific embodiment, the patient is a child with newly diagnosednon-brainstem high-grad glioma treated with RT with chemotherapy. Inanother specific embodiment, the patient is a child with newly diagnosednon-brainstem high-grad glioma treated with RT without chemotherapy. Inanother specific embodiment, the patient is a child with recurrentnon-brainstem high-grade glioma that has recurred after treatment.

In another embodiment, the brain cancer treated with the IL-13Rα2peptide-based vaccines described herein in accordance with the methodsdescribed herein is an astrocytoma. In a specific embodiment, the braincancer treated with the IL-13Rα2 peptide-based vaccines described hereinin accordance with the methods described herein is high-risk WHO GradeII Astrocytoma. In another specific embodiment, the brain cancer treatedwith the IL-13Rα2 peptide-based vaccines described herein in accordancewith the methods described herein is Oligo Astrocytoma. 6.7.3 PATIENTPOPULATION

In certain an IL-13Rα2 peptide-based vaccine or composition describedherein may be administered to a naïve subject, i.e., a subject that doesnot have brain cancer. In one embodiment, an IL-13Rα2 peptide-basedvaccine or composition described herein is administered to a naïvesubject that is at risk of acquiring brain cancer.

In certain embodiments, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a patient who has been diagnosedwith brain cancer. In some embodiments, an IL-13Rα2 peptide-basedvaccine or composition described herein is administered to a patientwith brain cancer before symptoms manifest or symptoms become severe. Ina preferred embodiment, the brain cancer is glioma.

In certain embodiments, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a patient who is in need oftreatment, prevention, and/or management of brain cancer. Such subjectsmay or may not have been previously treated for cancer or may be inremission, relapsed, or may have failed treatment. Such patients mayalso have abnormal cytogenetics. The 13Rα2 peptide-based vaccines andcompositions described herein may be used as any line of brain cancertherapy, e.g., a first line, second line, or third line of brain cancertherapy. In a specific embodiment, the subject to receive or receiving avaccine or pharmaceutical composition described herein is receiving orhas received other brain cancer therapies. In an alternative embodiment,the subject to receive or receiving a vaccine or pharmaceuticalcomposition described herein has not received or is not receiving otherbrain cancer therapies.

In a specific embodiment, the subject has been diagnosed with braincancer using techniques known to one of skill in the art including, butnot limited to, neurological examination; imaging methods (e.g.,computed tomography (CT), magnetic resonance imaging (MRI), ultrasound,X-ray imaging, fluid-attenuated inversion-recovery (FLAIR) sequences, T2weighted imaging, and positron emission tomography (PET) scans); andbiopsy (e.g., sterotactic biopsy). Tumor response to therapy may beevaluated by McDonald criteria or Response assessment in neuro-oncology(RANO) criteria. Tumor size or response to treatment can be evaluated byvarious magnetic resonance imaging techniques includingdiffusion-weighted imaging, perfusion-weighted imaging, dynamiccontrast-enhanced T1 permeability imaging, dynamic susceptibilitycontrast, diffusion-tensor imaging, and magnetic resonance spectroscopy,anatomic MRI T2-weighted images, fluid attenuated inversion recovery(FLAIR) T2-weighted images, and gadolinium-enhanced T1-weighted images.These imagining techniques can be used to assess tumor cellularity,white matter invasion, metabolic derangement including hypoxia andnecrosis, neovascular capillary blood volume, or permeability. Positronemission tomograph (PET) technology can also be used to image tumorresponse, such as 18F-fluoromisonidazole PET and3′-deoxy-3′-18F-fluorothymidine PET.

In one embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject that is undergoing or hasundergone radiation therapy to treat a brain cancer tumor. In a specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject concurrently or following radiationtherapy to treat a brain cancer tumor. In another embodiment, anIL-13Rα2 peptide-based vaccine or composition described herein isadministered to a subject before radiation therapy to treat a braincancer tumor and, in some embodiments, during and/or after the radiationtherapy. In some preferred embodiments, the radiation therapy isfractionated external beam radiotherapy, limited-field fractionatedexternal beam radiotherapy, whole brain radiotherapy, stereotacticradiosurgery, or craniospinal radiotherapy

In one embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject that is undergoing or hasundergone chemotherapy to treat a brain cancer tumor. In a specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject concurrently or followingchemotherapy to treat a brain cancer tumor. In another embodiment, anIL-13Rα2 peptide-based vaccine or composition described herein isadministered to a subject before chemotherapy to treat a brain cancertumor and, in some embodiments, during and/or after the chemotherapy. Insome prefered embodiments, the chemotherapy is temozolomide (Temodar®),nitrosurea, platinum-based regimens, etoposide, cisplatin, bevacizumab(Avastin®), irinotecan, cyclophosphamide, BCNU (carmustine),capecitabine, high-dose methotrexate, topotecan, high-dose ARA-C,hydroxyurea, α-inteferon, somatostatin analogue, intra-CSF chemotherapy(liposomal cytarabine, methotrexate, cytarabine, thiotepa, or rituximab(Rituxan®)).

In one embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject that has failed, isundergoing or has undergone more than one therapeutic strategy,including chemotherapy, radiation therapy, or surgery to treat a braincancer tumor. In a prefered embodiment, the brain cancer is glioma. Forexample, a patient may be failed, be undergoing, or have undergone bothchemotherapy and surgery. Alternatively, a patient may have undergone orbe undergoing both radiation and surgery. Moreover, a patient may haveundergone or be undergoing chemotherapy and radiation. In some preferedembodiments, the combined therapies that the patient failed, isundergoing, or has undergone are resection and temozolomide (Temodar®)(150-200 mg/m²) 5/28 schedule, resection and BCNU wafer (Gliader),bevacizumab (Avastin®) and chemotherapy, combination PCV (CCNU(lomustine) and procarbazine and vincristine), high-dose methotrexateand vincristine, procarbazine, cytaribine, or rituximab, high-dosechemotherapy with stem cell rescue, or rituximab (Rituxan®) andtemozolomide (Temodar®).

In one embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject that is undergoing or hasundergone surgery to remove a brain cancer tumor. In a specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject concurrently or following surgery toremove a brain cancer tumor. In another embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject before surgery to remove a brain cancer tumor and, in someembodiments, during and/or after surgery.

In certain embodiments, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject as an alternative toanother therapy, e.g., chemotherapy, radiation therapy, hormonaltherapy, surgery, small molecule therapy, anti-angiogenic therapy,and/or biological therapy including immunotherapy where the therapy hasproven or may prove too toxic, i.e., results in unacceptable orunbearable side effects for the subject.

In a specific embodiment, an IL-13Rα2 peptide-based vaccine orcomposition described herein is administered to subjects that will have,are undergoing, or have had radiation therapy. Among these subjects arethose that have received chemotherapy, hormonal therapy, small moleculetherapy, anti-angiogenic therapy, and/or biological therapy, includingimmunotherapy as well as those who have undergone surgery.

In another embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to subjects that will have, areundergoing, or have had hormonal therapy and/or biological therapy,including immunotherapy. Among these subjects are those that havereceived chemotherapy, small molecule therapy, anti-angiogenic therapy,and/or radiation therapy as well as those who have undergone surgery.

In certain embodiments, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject refractory to one or moretherapies. In one embodiment, that a cancer is refractory to a therapymeans that at least some significant portion of the cancer cells are notkilled or their cell division is not arrested. The determination ofwhether the cancer cells are refractory can be made either in vivo or invitro by any method known in the art for assaying the effectiveness of atherapy on cancer cells, using the art-accepted meanings of “refractory”in such a context. In various embodiments, a cancer is refractory wherethe amount of cancer cells has not been significantly reduced, or hasincreased.

In some embodiments, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject that is in remission frombrain cancer. In a specific embodiment, the subject has no detectablebrain cancer, i.e., no brain cancer is detectable using a conventionalmethod described herein (e.g., MM) or known to one of skill in the art.

In one embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject diagnosed with glioma. Ina specific embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject diagnosed with astrocytoma(e.g., pilocytic astrocytoma, diffuse astrocytoma, and anaplasticastrocytoma). In another specific embodiment, an IL-13Rα2 peptide-basedvaccine or composition described herein is administered to a subjectdiagnosed with glioblastoma. In another specific embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject diagnosed with oligodendroglioma. In another specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject diagnosed with brain stem glioma. Inanother specific embodiment, an IL-13Rα2 peptide-based vaccine orcomposition described herein is administered to a subject diagnosed withependymoma. In another specific embodiment, an IL-13Rα2 peptide-basedvaccine or composition described herein is administered to a subjectdiagnosed with a mixed tumor comprising more than one glial cell types.

In a specific embodiment, an IL-13Rα2 peptide-based vaccine orcomposition described herein is administered to a subject diagnosed withrecurrent malignant glioma. In another specific embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject diagnosed with high-risk WHO Grade II Astrocytomas. In anotherspecific embodiment, an IL-13Rα2 peptide-based vaccine or compositiondescribed herein is administered to a subject diagnosed with OligoAstrocytoma. In another specific embodiment, an IL-13Rα2 peptide-basedvaccine or composition described herein is administered to a subjectdiagnosed with recurrent WHO Grade II Glioma. In another specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject diagnosed with newly-diagnosedmalignant or intrinsic brain stem glioma. In another specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject diagnosed with incompletely resectednon-brainstem glioma. In another specific embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject diagnosed with recurrent unresectable low-grade glioma.

In a specific embodiment, an IL-13Rα2 peptide-based vaccine orcomposition described herein is administered to a subject diagnosed withacoustic schwannoma. In another specific embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject diagnosed with cranial pharyngioma. In another specificembodiment, an IL-13Rα2 peptide-based vaccine or composition describedherein is administered to a subject diagnosed with meningioma. Inanother specific embodiment, an IL-13Rα2 peptide-based vaccine orcomposition described herein is administered to a subject diagnosed withmedulloblastoma. In another specific embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject diagnosed with primary central nervous system lymphoma. Inanother specific embodiment, an IL-13Rα2 peptide-based vaccine orcomposition described herein is administered to a subject diagnosed witha tumor of the pineal gland (e.g., a pineal astrocytic tumor or a pinealparenchymal tumor). In another specific embodiment, an IL-13Rα2peptide-based vaccine or composition described herein is administered toa subject diagnosed with a tumor of the pituitary gland.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein is a human adult.In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein is an elderlyhuman subject. In certain embodiments, a subject to be administered anIL-13Rα2 peptide-based vaccine or composition described herein is ahuman child. In certain embodiments, a subject to be administered anIL-13Rα2 peptide-based vaccine or composition described herein is ahuman infant. In certain embodiments, a subject to be administered anIL-13Rα2 peptide-based vaccine or composition described herein is ahuman toddler.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein is HLA-A2 positiveas determined by, e.g., flow cytometry.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a Karnofskyperformance status (KPS) of >60. The KPS is used as a stratification andselection variable in randomized trials of chemotherapeutic agents andhas a range of 0-100. Patients with a score >60 are unable to work, areable to live at home, and can care for most of their personal needs withvarying degrees of required assistance. Patients with a score >70 carryon normal activity with effort and show some signs and symptoms of thedisease. Patients with a score >80 are able to carry on normal activityand only show minor signs or symptoms of the disease. Patients with ascore >90 are normal, have no health complaints, and show no signs orsymptoms of the disease.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a white bloodcount of about 1000/mm³, 1500/mm³, 2000/mm³, 2500/mm³, 3000/mm³, or3500/mm³ or about 1000/mm³ to 1500/mm³, 1000/mm³ to 2000/mm³, 1500/mm³to 2500/mm³, 1500/mm³ to 3000/mm³, 2000/mm³ to 3500/mm³, or 2500/mm³ to3500/mm³. In a specific embodiment, a subject to be administered anIL-13Rα2 peptide-based vaccine or composition described herein has awhite blood count greater than or equal to 2500/mm³.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a lymphocytecount of about 100/mm³, 200/mm³, 300/mm³, 400/mm³, 500/mm³, or 600/mm³or about 100/mm³ to 400/mm³, 200/mm³ to 400/mm³, 300/mm³ to 500/mm³,300/mm³ to 600/mm³, 400/mm³ to 500/mm³, or 400/mm³ to 600/mm³. In aspecific embodiment, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a lymphocytecount greater than or equal to 400/mm³.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a plateletcount of about 25,000/mm³, 50,000/mm³, 75,000/mm³, 100,000/mm³,200,000/mm³, or 300,000/mm³ or about 25,000/mm³ to 100,000/mm³,50,000/mm³ to 100,000/mm³, 75,000/mm³ to 100,000/mm³, 100,000/mm³ to200,000/mm³, 100,000/mm³ to 300,000/mm³, or 200,000/mm³ to 300,000/mm³.In a specific embodiment, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a plateletcount greater than or equal to 100,000/mm³.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a hemoglobincount of about 5 g/dL, 10 g/dL, 15 g/dL, or 20 g/dL, or about 5 to 10g/dL, 5 to 15 g/dL, 10 to 15 g/dL, or 10 to 20 g/dL. In a specificembodiment, a subject to be administered an IL-13Rα2 peptide-basedvaccine or composition described herein has a hemoglobin count greaterthan or equal to 10 g/dL.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has AST, ALT, GGT,LDH, and alkaline phosphatase levels within 1, 1.5, 2, 2.5, or 3 timesthe upper normal limit. In a specific embodiment, a subject to beadministered an IL-13Rα2 peptide-based vaccine or composition describedherein has AST, ALT, GGT, LDH, and alkaline phosphatase levels within2.5 times the upper normal limit.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has a totalbilrubin of about 1 mg/dL, 1.5 mg/dL, 2 mg/dL, 2.5 mg/dL, or 3 mg/dL, orabout 1.5 to 2.5 mg/dL, 1.5 to 3 mg/dL, 2 to 2.5 mg/dL, or 2 to 3 mg/dL.In a specific embodiment, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has total bilrubingreater than or equal to 2 mg/dL.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has serumcreatinine levels within 0.5, 1, 1.5, 2, 2.5, or 3 times the uppernormal limit. In a specific embodiment, a subject to be administered anIL-13Rα2 peptide-based vaccine or composition described herein has serumcreatinine levels within 1.5 times the upper normal limit.

In certain embodiments, a subject to be administered an IL-13Rα2peptide-based vaccine or composition described herein has coagulationtests PT and PTT that are within 0.5, 1, 1.5, 2, 2.5, or 3 times thenormal limits. In certain embodiments, a subject to be administered anIL-13Rα2 peptide-based vaccine or composition described herein hascoagulation tests PT and PTT that are within normal limits.

In some embodiments, it may be advisable not to administer an IL-13Rα2peptide-based vaccine or composition described herein to one or more ofthe following patient populations: elderly humans; infants younger than6 months old; pregnant individuals; infants under the age of 1 yearsold; children under the age of 2 years old; children under the age of 3years old; children under the age of 4 years old; children under the ageof 5 years old; adults under the age of 20 years old; adults under theage of 25 years old; adults under the age of 30 years old; adults underthe age of 35 years old; adults under the age of 40 years old; adultsunder the age of 45 years old; adults under the age of 50 years old;elderly humans over the age of 70 years old; elderly humans over the ageof 75 years old; elderly humans over the age of 80 years old; elderlyhumans over the age of 85 years old; elderly humans over the age of 90years old; elderly humans over the age of 95 years old; subjectsundergoinging chemotherapy; subjects undergoing radiation therapy;subjects undergoing biologic therapy; subjects undergoing interferontherapy; subjects that receive allergy desensitization injections;subjects that take illicit drugs; subjects receiving growth factortreatments (e.g. Procrit®, Aranesp®, Neulasta®); subjects receivinginterleukin treatments (e.g. Proleukin®); subjects with metastaticdisease; female subjects that are breast-feeding; subjects with activeviral, bacterial, or fungal infection; subjects with a history ofpresence of autoimmune disease; subject with HIV; subjects being treatedwith investigational medicines that do not comprise a vaccine describedherein; subjects with gliomatosis cerebri, cranial or spinalleptomeningeal metastatic disease; and/or subjects undergoingimmunosuppressive treatment.

6.7.4 Combination Therapies

In certain embodiments, the methods provided herein for preventing,treating, and/or managing brain cancer comprise administering to apatient (e.g., a human patient) in need thereof a prophylacticallyand/or a therapeutically effective regimen, the regimen comprisingadministering to the patient an IL-13Rα2 peptide-based vaccine orcomposition described herein and one or more additional therapies, saidadditional therapy not being an IL-13Rα2 peptide-based vaccine orcomposition described herein. The an IL-13Rα2 peptide-based vaccine orcomposition described herein and the additional therapy can beadministered separately, concurrently, or sequentially. The combinationtherapies can act additively or synergistically.

The combination therapies can be administered to a subject in the samepharmaceutical composition. Alternatively, the combination therapies canbe administered concurrently to a subject in separate pharmaceuticalcompositions. The combination therapies may be administered to a subjectby the same or different routes of administration.

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, or is currently being used for the prevention, treatment,and/or management of cancer (e.g., brain cancer) can be used incombination with an IL-13Rα2 peptide-based vaccine or compositiondescribed herein in the methods described herein. Therapies include, butare not limited to, peptides, polypeptides, antibodies, conjugates,nucleic acid molecules, small molecules, mimetic agents, syntheticdrugs, inorganic molecules, and organic molecules. Non-limiting examplesof cancer therapies include chemotherapy, radiation therapy, hormonaltherapy, surgery, small molecule therapy, anti-angiogenic therapy,differentiation therapy, epigenetic therapy, radioimmunotherapy,targeted therapy, and/or biological therapy including immunotherapy. Incertain embodiments, a prophylactically and/or therapeutically effectiveregimen of the invention comprises the administration of a combinationof therapies.

In one embodiment, the prior chemotherapy is temolozimide. Inembodiment, the prior therapy is radiation therapy. In anotherembodiment, the prior therapy is a combination of temozolomide andradiation therapy. In a preferred embodiment, the combination oftemozolomide and radiation are administered using the Stupp regimen. Inanother embodiment, the prior therapy is surgery. In some embodiments,the patient undergoes surgery before the initiation of combinationtherapy. In some embodiments, the patient undergoes surgery beforetreatment with temozolomide. In some embodiments, the patient undergoessurgery before the initiation of radiation therapy. In each of theseembodiments that describe the use of combination therapy, the IL-13Rα2peptide-based vaccine may be administered before, during, or after thetreatment of the patient with the therapy that is being combined.

Examples of cancer therapies which can be used in combination with anIL-13Rα2 peptide-based vaccine or composition described herein include,but are not limited to: acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthracyclin;anthramycin; asparaginase; asperlin; azacitidine (Vidaza); azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bisphosphonates (e.g., pamidronate(Aredria), sodium clondronate (Bonefos), zoledronic acid (Zometa),alendronate (Fosamax), etidronate, ibandornate, cimadronate,risedromate, and tiludromate); bizelesin; bleomycin sulfate; brequinarsodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide;carbetimer; carboplatin; carmustine; carubicin hydrochloride;carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin;cladribine; crisnatol mesylate; cyclophosphamide; cytarabine (Ara-C);dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine(Dacogen); demethylation agents, dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; EphA2 inhibitors; elsamitrucin;enloplatin; enpromate; epipropidine; epirubicin hydrochloride;erbulozole; esorubicin hydrochloride; estramustine; estramustinephosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine;fadrozole hydrochloride; fazarabine; fenretinide; floxuridine;fludarabine phosphate; fluorouracil; flurocitabine; fosquidone;fostriecin sodium; gemcitabine; histone deacetylase inhibitors (HDACs)gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;ifosfamide; ilmofosine; imatinib mesylate (Gleevec, Glivec); interleukinII (including recombinant interleukin II, or rIL2), interferon alpha-2a;interferon alpha-2b; interferon alpha-n1; interferon alpha-n3;interferon beta-I a; interferon gamma-I b; iproplatin; irinotecanhydrochloride; lanreotide acetate; lenalidomide (Revlimid); letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; anti-CD2 antibodies (e.g., siplizumab(MedImmune Inc.; International Publication No. WO 02/098370, which isincorporated herein by reference in its entirety)); megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;nogalamycin; ormaplatin; oxaliplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other examples of cancer therapies which can be used in combination withan IL-13Rα2 peptide-based vaccine or composition described hereininclude, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, dioxamycin; diphenyl spiromustine; docetaxel; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; HMG CoAreductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin,lescol, lupitor, lovastatin, rosuvastatin, and simvastatin); hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; LFA-3TIP(Biogen, Cambridge, Mass.; International Publication No. WO 93/0686 andU.S. Pat. No. 6,162,432); liarozole; linear polyamine analogue;lipophilic disaccharide peptide; lipophilic platinum compounds;lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinaseinhibitors; menogaril; merbarone; meterelin; methioninase;metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim;mismatched double stranded RNA; mitoguazone; mitolactol; mitomycinanalogues; mitonafide; mitotoxin fibroblast growth factor-saporin;mitoxantrone; mofarotene; molgramostim; monoclonal antibody, humanchorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wallsk; mopidamol; multiple drug resistance gene inhibitor; multiple tumorsuppressor 1-based therapy; mustard anticancer agent; mycaperoxide B;mycobacterial cell wall extract; myriaporone; N-acetyldinaline;N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine;napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronicacid; neutral endopeptidase; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; thalidomide; velaresol; veramine; verdins; verteporfin;vinorelbine; vinxaltine; VITAXIN™ (see U.S. Patent Pub. No. US2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods of Preventing orTreating Inflammatory or Autoimmune Disorders by Administering Integrinαvβ3 Antagonists in Combination With Other Prophylactic or TherapeuticAgents”); vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

In some embodiments, the therapy(ies) used in combination with anIL-13Rα2 peptide-based vaccine or composition described herein is animmunomodulatory agent. Non-limiting examples of immunomodulatory agentswhich can be used in combination with an IL-13Rα2 peptide-based vaccineor composition described herein include proteinaceous agents such ascytokines, peptide mimetics, and antibodies (e.g., human, humanized,chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments orepitope binding fragments), nucleic acid molecules (e.g., antisensenucleic acid molecules and triple helices), small molecules, organiccompounds, and inorganic compounds. In particular, immunomodulatoryagents include, but are not limited to, methotrexate, leflunomide,cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline,azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone(MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin(sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,cytokine receptor modulators, and modulators mast cell modulators. Otherexamples of immunomodulatory agents can be found, e.g., in U.S.Publication No. 2005/0002934 A1 at paragraphs 259-275 which isincorporated herein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent.

In some embodiments, the therapy(ies) used in combination with a anIL-13Rα2 peptide-based vaccine or composition described herein is ananti-angiogenic agent. Non-limiting examples of anti-angiogenic agentswhich can be used in combination with an IL-13Rα2 peptide-based vaccineor composition described herein include proteins, polypeptides,peptides, conjugates, antibodies (e.g., human, humanized, chimeric,monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2 fragments, andantigen-binding fragments thereof) such as antibodies that specificallybind to TNF-α, nucleic acid molecules (e.g., antisense molecules ortriple helices), organic molecules, inorganic molecules, and smallmolecules that reduce or inhibit angiogenesis. Other examples ofanti-angiogenic agents can be found, e.g., in U.S. Publication No.2005/0002934 A1 at paragraphs 277-282, which is incorporated byreference in its entirety. In a prefered embodiment, the anti-angiogenictherapy is bevacizumab (Avastin®). In other embodiments, thetherapy(ies) used in accordance with the invention is not ananti-angiogenic agent.

In some embodiments, the therapy(ies) used in combination with anIL-13Rα2 peptide-based vaccine or composition described herein is ananti-inflammatory agent. Non-limiting examples of anti-inflammatoryagents which can be used in combination with an IL-13Rα2 peptide-basedvaccine or composition described herein include any anti-inflammatoryagent, including agents useful in therapies for inflammatory disorders,well-known to one of skill in the art. Non-limiting examples ofanti-inflammatory agents include non-steroidal anti-inflammatory drugs(NSAIDs), steroidal anti-inflammatory drugs, anticholinergics (e.g.,atropine sulfate, atropine methylnitrate, and ipratropium bromide(ATROVENT™)), beta2-agonists (e.g., abuterol (VENTOLIN™ and PROVENTIL™),bitolterol (TORNALATE™), levalbuterol (XOPONEX™), metaproterenol(ALUPENT™), pirbuterol (MAXAIR™), terbutlaine (BRETHAIRE™ andBRETHINE™), albuterol (PROVENTIL™, REPETABS™, and VOLMAX™), formoterol(FORADIL AEROLIZER™), and salmeterol (SEREVENT™ and SEREVENT DISKUS™)),and methylxanthines (e.g., theophylline (UNIPHYL™, THEO-DUR™, SLO-BID™,AND TEHO-42™)). Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™ NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxgenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), corticosteroids (e.g., methylprednisolone (MEDROL™)),cortisone, hydrocortisone, prednisone (PREDNISONE™ and DELTASONE™),prednisolone (PRELONE™ and PEDIAPRED™), triamcinolone, azulfidine, andinhibitors of eicosanoids (e.g., prostaglandins, thromboxanes, andleukotrienes. Other examples of anti-inflammatory agents can be found,e.g., in U.S. Publication No. 005/0002934 A1 at paragraphs 290-294,which is incorporated by reference in its entirety. In otherembodiments, the therapy(ies) used in accordance with the invention isnot an anti-inflammatory agent.

In certain embodiments, the therapy(ies) used in combination with anIL-13Rα2 peptide-based vaccine or composition described herein is analkylating agent, a nitrosourea, an antimetabolite, and anthracyclin, atopoisomerase II inhibitor, or a mitotic inhibitor. Alkylating agentsinclude, but are not limited to, busulfan, cisplatin, carboplatin,cholormbucil, cyclophosphamide, ifosfamide, decarbazine,mechlorethamine, melphalan, and temozolomide. Nitrosoureas include, butare not limited to carmustine (BCNU) and lomustine (CCNU).Antimetabolites include but are not limited to 5-fluorouracil,capecitabine, methotrexate, gemcitabine, cytarabine, and fludarabine.Anthracyclins include but are not limited to daunorubicin, doxorubicin,epirubicin, idarubicin, and mitoxantrone. Topoisomerase II inhibitorsinclude, but are not limited to, topotecan, irinotecan, etopiside(VP-16), and teniposide. Mitotic inhibitors include, but are not limitedto taxanes (paclitaxel, docetaxel), and the vinca alkaloids(vinblastine, vincristine, and vinorelbine).

Currently available cancer therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006). In accordance with the present invention, the dosages andfrequency of administration of chemotherapeutic agents are describedsupra.

6.7.5 Biological Assays

The IL-13Rα2 peptide-based vaccines and compositions described hereincan be tested for their ability to treat, prevent, or manage braincancer.

6.7.5.1 In Vivo Assays

The IL-13Rα2 peptide-based vaccines and compositions described hereincan be tested in suitable animal model systems prior to use in humans.Such animal model systems include, but are not limited to, rats, mice,chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal systemwell-known in the art may be used. Several aspects of the procedure mayvary; said aspects include, but are not limited to, the temporal regimeof administering the vaccine components, whether such vaccine componentsare administered separately or as an admixture, and the frequency ofadministration of the vaccine components.

Animal models for cancer can be used to assess the efficacy of anIL-13Rα2 peptide-based vaccine or composition described herein or acombination therapy described herein. Examples of animal models forbrain cancer include, but are not limited to, xenograft studies usingbrain cancer cell lines that express IL-13Rα2, or primary human tumorcells that express IL-13Rα2. In these models, mice are immunized toinduce an IL-13Rα2-specific T cell response, which is then evaluated forits ability to inhibit the growth of the tumor. In one embodiment, thetumor xenograft forms prior to the immunization to test the ability ofthe IL-13Rα2-specific T cell response to inhibit the growth of thepreexisting tumor. In another embodiment, the IL-13Rα2-specific T cellresponse is induced prior to the injection of the tumor cells, toevaluate the ability of the immune response to prevent the formation ofa tumor.

6.7.5.2 Cytotoxicity Assays

The toxicity and/or efficacy of the IL-13Rα2 peptide-based vaccines andcompositions described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Therapeutic regimens that exhibit large therapeutic indices arepreferred. While therapeutic regimens that exhibit toxic side effectsmay be used, care should be taken to design a delivery system thattargets such agents to the site of affected tissue in order to minimizepotential damage to uninfected cells and, thereby, reduce side effects.

6.8 Articles of Manufacture

Also encompassed herein is a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. Thepharmaceutical product may contain, for example, the components of anIL-13Rα2 peptide-based vaccine described herein in a unit dosage form.

In a specific embodiment, the unit dosage form is suitable forparenteral, intravenous, intramuscular, intranasal, or subcutaneousdelivery. Thus, encompassed herein are solutions, preferably sterile,suitable for each delivery route.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products provided herein include instructions foruse or other informational material that advise the physician,technician, or patient on how to appropriately prevent or treat braincancer in question. In other words, the article of manufacture includesinstruction means indicating or suggesting a dosing regimen including,but not limited to, actual doses, monitoring procedures, and otherinformation.

Specifically, provided herein is an article of manufacture comprisingpackaging material, such as a box, bottle, tube, vial, container,sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; andat least one unit dosage form of a vaccine or pharmaceutical compositiondescribed herein contained within said packaging material, wherein saidvaccine or pharmaceutical composition described herein comprises anIL-13Rα2 peptide-based vaccine described herein, and wherein saidpackaging material includes instruction means which indicate that saidIL-13Rα2 peptide-based vaccine described herein can be used to prevent,manage, and/or treat brain cancer or one or more symptoms thereof byadministering specific doses and using specific dosing regimens asdescribed herein.

7. EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

7.1 Example 1

This example demonstrates the identification of modified peptides forIL-13Rα2₃₄₅₋₃₅₃ that enhance induction of the CTL response againstnative IL-13Rα2₃₄₅₋₃₅₃.

Three modified peptides were synthesized as listed in Table 1. Thebinding capability of these modified peptides was assessed using anHLA-A2 transfected T2 cell line. Aliquots of T2 cells were incubatedwith modified peptides or IL-13Rα2₃₄₅₋₃₅₃ at 1 nM overnight, and thenexamined for the surface expression levels of HLA-A2 on T2 cells by flowcytometry. Since stable binding of HLA-A2 with peptide epitopes furtherstabilizes the surface expression of HLA-A2 (Francini et al., 2002;Alves et al., 2003), quantitative expression levels of HLA-A2, which isindicated by Mean Fluorescence Intensity (MFI) in Table 1, correlatewith the binding affinity of the peptide-epitopes that are co-incubatedwith the T2 cells. The modified peptides V9 and A1V9 possess higherbinging affinity to HLA-A2 than the native IL-13Rα2₃₄₅₋₃₅₃ (Table 1),suggesting the possibility that these modified peptides are moreimmunogenic than the IL-13Rα2₃₄₅₋₃₅₃.

TABLE 1 Binding Amino Acid Index Peptide Sequence (MFI*) DesignationNative WLPFGFILI 237.4 Native IL-13Rα₃₄₅₋₃₅₃ (SEQ ID NO: 1) V9: I wasWLPFGFILV 375.6 V9 replaced with (SEQ ID NO: 2) V at P9 A1V9: W→A at P1,ALPFGFILV 462.8 A1V9 and I→V at P9 (SEQ ID NO: 3) A1V9: W→E at P1,ELPFGFILV 241.6 E1V9 and I→V at P9 (SEQ ID NO: 4) Control — 121.8 —(Non-Peptide) *Mean Fluorescence Intensity at the peptide concentrationof 1 nM

7.2 Example 2

This example demonstrates that CTL induced by the agonist analogue V9recognized peptide IL-13Rα2₃₄₅₋₃₅₃ presented on HLA-A*0201 moreefficiently than CTL induced by the wild type peptide.

Dendritic cells (DCs) derived from HLA-A*0201+ glioma patients werepulsed with either V9, A1V9, E1V9, a control influenza (flu), or thewild type peptide (10 μg/ml), and used to stimulate autologous CD8+ Tcells. On day 7, the individual responder cell cultures were thenrestimulated once with autologous DCs loaded with the correspondingpeptide used in the primary stimulation. Specific CTL activity of theinduced T cell lines was first tested with T2 cells loaded with the wildtype IL-13Rα2₃₄₅₋₃₅₃, or no peptide on day 10.

As depicted in FIG. 1, the T cells that had been stimulated with eitherwild type (IL-13R) or agonist analogues (V9, A1V9 and E1V9) efficientlylysed T2 target cells pulsed with 100 ng/ml wild type IL-13Rα2₃₄₅₋₃₅₃;whereas only low background lysis was observed in the absence of thepeptide on T2 cells. T cells that had been stimulated with the controlflu-peptide or no-peptide (control) did not demonstrate any lyticactivity over background levels. These results demonstrated that the CTLlines induced with the wild type or agonist analogues recognized andlysed the cells presenting wild type IL-13Rα2₃₄₅₋₃₅₃ epitopespecifically. In particular, the V9 peptide induced a significantlyhigher level of antigen-specific CTL response in comparison to the wildtype IL-13Rα2₃₄₅₋₃₅₃ at each effector/target (E/T) ratio (p=0.018, 0.020and 0.011 at an E/T ratio of 50, 25 and 12.5, respectively). The sameset of experiments were repeated with at least three individual HLA-A2+glioma patients, and the V9 peptide consistently demonstrated higher CTLactivities than the native IL-13Rα2₃₄₅₋₃₅₃ in all four donors tested(data not shown).

Subsequently, the sensitivity of the CTL lines induced by agonistanalogues or the wild type peptide was examined with T2 cells loadedwith various concentrations (1-100 nM) of the IL-13Rα2₃₄₅₋₃₅₃ peptide by4-Hr ⁵¹Cr-release assay (FIG. 2). All CTL lines demonstratedpeptide-dose dependent lytic activities against peptide-loaded T2 cells.The CTL line induced by the agonist analogue V9 demonstrated higher CTLactivities than the wild type IL-13Rα2₃₄₅₋₃₅₃ at allpeptide-concentrations examined (P=0.029, 0.039 and 0.018 at 1, 10 and100 nM, respectively). It is noteworthy that the average percent lysisvalue achieved by V9-induced CTL with 1 nM IL-13Rα2₃₄₅₋₃₅₃ was higherthan that demonstrated with wild type peptide-induced CTL with 100 nMpeptide, although this did not demonstrate a statistical significancedue to a large standard variation. These results indicate that the V9peptide is more efficient than the wild type peptide in inducing CTLthat are capable of recognizing low concentrations of the target wildtype IL-13Rα2₃₄₅₋₃₅₃ peptide. This ability is important because humantumor cells express low levels of target CTL epitopes on theirHLA-molecules (Bakker et al., 1995; Lupetti et al., 1998).

7.3 Example 3

This example demonstrates that CTL induced by modified peptides lysedHLA-A2+ glioma cells that express IL-13Rα2 more efficiently than CTLinduced by the native peptide.

The ability of modified peptides, such as IL-13Rα2-V9, to enhance theCTL activity against HLA-A2+ human glioma cells that endogenouslyexpressed and presented IL-13Rα2-derived epitopes was examined. Humanglioma cell lines U251 and SNB19 express HLA-A2 and IL-13Rα2, whereashuman glioma cell line A172 expresses IL-13Rα2 but not HLA-A2 (Okano etal., 2002). Therefore, U251 and SNB19 were used as relevant targetglioma cells, while A172 served as a negative control line todemonstrate HLA-A2-restriction of the response.

The lytic ability of the peptide-induced CTL lines against these gliomacells was examined using 4-hr ⁵¹Cr-release assays. As illustrated inFIG. 3, the U-251 and SNB19 cell lines were highly susceptible tocytotoxic activity of all the CTL lines that had been induced withIL-13Rα2₃₄₅₋₃₅₃ or each of its modified peptides. A172 cells, incontrast, were not lysed beyond the background level (<10%) by any ofthe CTL lines tested, suggesting that the IL-13Rα2₃₄₅₋₃₅₃ or modifiedpeptide-induced CTL lines lysed SNB19 and U-251 glioma cells in anHLA-A2 restricted manner (data not shown). The T cells stimulated with amelanoma associated antigen epitope Mart-127-35 and T cells with nopeptide stimulation showed only background level (<10%) lysis at allEffector/Target (E/T) ratios tested (data not shown). In this particularpatient, both IL-13Rα2-V9 and -A1V9 induced higher levels of lysis ofSNB19 and U-251 in each E/T ratio in comparison to the nativeIL-13Rα2₃₄₅₋₃₅₃ peptide.

To determine the specificity of the lytic activity, cold targetcompetition experiments were performed by addition of non-radiolabeled(cold) T2 cells pulsed with IL-13Rα2₃₄₅₋₃₅₃ peptide in the 4-h⁵¹Cr-release assay (FIG. 4). The anti-SNB19 glioma cell lytic activitiesby the CTL lines induced by the native IL-13Rα2₃₄₅₋₃₅₃ or IL-13Rα2-V9were almost completely inhibited by the addition of the cold T2 cellspulsed IL-13Rα2₃₄₅₋₃₅₃. The CTL activities, however, were not inhibitedby the addition of non-peptide pulsed cold T2 cells, demonstrating thatthe lytic ability of the CTLs was specific for the epitopeIL-13Rα2₃₄₅₋₃₅₃.

Furthermore, anti-HLA-A2 antibody (W6/32) was used to block the HLA-A2mediated signaling in the CTL reactivity. As illustrated in FIG. 5,addition of this antibody inhibited the CTL-mediated lysis, confirmingthat the anti-glioma CTL reactivity induced by these peptides was HLA-A2restricted.

7.4 Example 4

This example demonstrates the vaccination of HLA-A2 transgenic (HHD)mice with IL-13Rα2-derived CTL epitopes.

In order to examine whether immunization with IL-13Rα2₃₄₅₋₃₅₃ and/or itsmodified peptides can elicit CTL responses in vivo, and also to examinewhether induced CTL responses can mediate therapeutic anti-tumorresponses against IL-13Rα2₃₄₅₋₃₅₃-expressing brain tumors, the HHD micewere obtained from Dr. Francois A. Lemonnier (Pasteur Institute, Paris).HHD mice are D^(b)×β2 microglobulin (02M) null, and transgenic formodified HLA-A2.1132 microglobulin single chain (HHD gene) (Pascolo etal., 1997). In vivo experiments showed that HHD mice exhibitHLA-A2-restricted responses to multiepitope proteins such as intactinfluenza virus (Pascolo et al., 1997) and novel cancer associatedantigens, such as EphA2 (Alves et al., 2003), HER-2/neu and hTERT(Scardino et al., 2002), MAGE (Graff-Dubois et al., 2002) and a novelbreast carcinoma associated BA46 (Carmon et al., 2002). Hence, thesemice are a useful tool for the identification and characterization ofpotential tumor-derived, HLA-A2-restricted CTL epitopes.

To create an HHD mouse-syngeneic tumor cell line that expressesIL-13Rα2, HHD gene-transfected EL4 lymphoma cells (EL4-HHD) wereobtained. EL4-HHD cells have been generated from EL4 by depletion ofD^(b)×β2M and insertion of modified HLA-A2.1-β2M single chain (Pascoloet al., 1997), thereby allowing syngeneic transplantation in HHD mice.EL4-HHD cells were stably transfected with an expression plasmidencoding IL-13Rα2. The cell line (EL4-HHD-IL-13Rα2) expressed IL-13Rα2protein and formed tumors both in subcutaneous (s.c.) and intracranial(i.c.) space following injections to syngeneic HHD mice.

7.5 Example 5

This example demonstrates that in vivo immunization of HHD mice with themodified peptides induced higher magnitudes of CTL responses than thenative peptide against the target cells expressing IL-13Rα2₃₄₅₋₃₅₃.

HHD mice received (on days 7 and 14) s.c. injections of 100 μg ofpeptide IL-13Rα2-V9, -A1V9, IL-13Rα2₃₄₅₋₃₅₃, or MART-127-35 emulsifiedin incomplete Freund's adjuvant (IFA) in the presence of 140 μg of theI-A^(b)-restricted HBV core₁₂₈₋₁₄₀ (TPPAYRPPNAPIL) (SEQ ID NO:5)T-helper epitope, which stimulates a CD4+ helper T cell response,thereby promoting the stimulation of CD8+ CTLs. Control animals receivedIFA containing HBV helper-peptide only. Eleven days after the lastimmunization, the animals were sacrificed, and 5×10⁶ spleen cells (SPCs)were stimulated in vitro with the same peptide that was used for in vivostimulation (10 On day 6 of culture, the bulk populations were testedfor specific cytotoxicity against the EL4-HHD cells expressing IL-13Rα2or EL4-HHD pulsed with IL-13 Rα2₃₄₅₋₃₅₃.

EL4-HHD-IL-13Rα2 and EL4-HHD were labeled with 100 μCi of ⁵¹Cr for 60min, plated in 96-well V-bottomed plates (3×10³ cell/well). LabeledEL4-HHD were pulsed with IL-13Rα2₃₄₅₋₃₅₃ (1 μM) at 37° C. for 2 h.Control target cells were pulsed with no peptides. Stimulated SPCs werethen added as effector cells and incubated at 37° C. for 4 h. Onehundred μl of supernatant were collected and radioactivity measured in agamma counter.

FIG. 6 demonstrates that the CTL responses induced by the modifiedpeptides were able to lyse T2 cells loaded with the nativeIL-13Rα2₃₄₅₋₃₅₃. Control non-pulsed EL4-HHD cells were not lysed by theCTLs beyond background levels (shown in FIG. 7). Furthermore, theimmunization with IL-13Rα2-V9 displayed a trend toward higher levels ofCTL reactivity against the EL4-HHD cells pulsed with the nativeIL-13Rα2₃₄₅₋₃₅₃ peptide than other peptides examined, although thedifference was not statistically significant due to the variation withinthe triplicated samples. These data support the previous set of datawith human HLA-A2+ patient derived T cells, in which the modifiedpeptides induced higher levels of anti-IL-13Rα2₃₄₅₋₃₅₃ CTL response thanthe native peptide.

The ability of the same HHD mice-derived CTLs used in FIG. 6 to lyseEL4-HHD-IL-13Rα2 cells was examined in order to evaluate the ability ofthe CTLs to recognize the IL-13Rα2₃₄₅₋₃₅₃ peptide that is naturallyprocessed by cells that endogenously express IL-13Rα2. FIG. 7illustrates that immunization with the IL-13Rα2₃₄₅₋₃₅₃, IL-13Rα2-V9 or-A1V9 induced a specific CTL activity against EL4-HHD-IL-13Rα2 cells.The CTL activities were antigen-specific because control EL4-HHD werenot lysed beyond the background level. Modified peptides IL-13Rα2-V9 and-A1V9 induced higher magnitude of CTL activities in comparison to nativeIL-13Rα2₃₄₅₋₃₅₃ against the EL4-HHD-IL-13Rα cells (p<0.05 at alleffector/target ratios). The in vivo anti-tumor effect of vaccinationswith the IL-13Rα2₃₄₅₋₃₅₃ or modified IL-13Rα2 peptides in HHD micebearing EL4-HHD-IL-13Rα2 tumors is currently being evaluated.

7.6 Example 6

This example demonstrates that EphA2 has available HLA-A2-restricted CTLepitopes.

EphA2 is an attractive tumor-associated antigen and a target fortumor-vaccines, as 5 HLA-A2 and 3 DR4 T cell epitopes have beenpreviously identified (Tatsumi et al., 2003). As shown in FIG. 8, 9 of14 human glioblastoma multiforme (GBM) and 6 of 9 anaplastic astrocytoma(AA) cases express high levels of EphA2. In addition, anti-glioma CTLreactivity has been induced in CD8+ cells obtained from HLA-A2+ gliomapatients by stimulation with the EphA2883-891 epitope (FIG. 9). Thisresponse was specific for the EphA2883-891 epitope because the parallelassay using T2 cells loaded with EphA2883-891 demonstrated apeptide-specific response in comparison to the control unloaded T2target (not shown). These data strongly suggest that EphA2883-891 canserve as a CTL epitope.

7.7 Example 7

This Example describes a phase I/II trial performed to evaluate thesafety and immunogenicity of a novel vaccination with α-type-1-polarizeddendritic cells (αDC1) loaded with synthetic peptides for gliomaassociated antigen (GAA) epitopes and administration of poly-ICLC inhuman leukocyte antigen (HLA)-A2⁺ patients with recurrent malignantgliomas. GAAs for these peptides are EphA2, interleukin-13 receptor(IL-13R) α2, YKL-40 and gp100.

7.7.1 Patients and Methods

7.7.1.1 Patients

Patients with recurrent malignant glioma were enrolled with informedconsent and approvals by the institutional review board (IRB) and USFood and Drug Administration (FDA) (BB-IND #12415). Clinicalcharacteristics of patients are summarized in Tables 2 and 3A.Enrollment criteria included: histological diagnosis of glioblastomamultiforme (GBM) or anaplastic glioma (AG) including anaplasticastrocytoma (AA), anaplastic oligodendroglioma (AO) or anaplasticoligoastrocytoma (AOA); up to 2 previous recurrences; >18 years old;Karnofsky performance status >60; adequate liver and renal function andHLA-A2+. Minimum doses of corticosteroid (dexamethasone up to 4 mg/day)were permitted. Twenty-two patients were enrolled and received at leastone vaccination. Nineteen of 22 patients completed the scheduled initial4 immunizations; three patients (Patients 4, 11 and 13) were withdrawnfrom the protocol due to early tumor progression. Nine patientscompleted 5 additional booster vaccinations. Immunologic and safety dataare presented on patients who had at least 4 vaccinations (n=19), and atleast one vaccination (n=22), respectively.

TABLE 2 Demographics and Clinical Characteristics of ParticipatingPatients Total DC Dose Level (n = 22) (No. of DC/dose) No. ofCharacteristics 1 (1 × 10⁷) 2 (3 × 10⁷) Patients % Received at least onevaccine 11 11  22 Completed at least 4 vaccines 10 9 19 86 Female(received at least 4 5 4 9 47 vaccines) Median age, years 52 46 48 Range37-71 28-63 28-71 Tumor Histology AA 3 2 5 23 AO 1 2 3 14 AOA 1 0 1 4GBM 6 7 13 59 No. of Previous Recurrences 0 7 4 11 50 1 2 5 7 32 2 2 2 418

TABLE 3A Age/ No. DC ELISPOT Pt Gen- Tumor Location of Prior Prev. IL-12(pos/neg) by Week 9 Tetramer RR at TTP OS ID der Histol. Tumor TherapyRec. (pg) I E Y G Pa I E G week 9 (Mo) (Mo) Dose 1 57/M GBM Rt. Temp/PaRes/RT/TMZ/Mol 1 10 N N N N N P P P PR 7 14 Level 1 2 52/M GBM Rt.Temporal Res/RT/TMZ 1 25 P N N P N P P P PD <2 12 (1 × 10⁷ 3 37/M AA Rt.Parietal Resx2/RT/ 2 25 N N N N N N N N SD 5 10 DC/ TMZ/Mol dose) 4 68/FAA Rt. Frontal SB/RT/TMZ 0 <10 Not Tested Not Tested  ND* <2 >37 5 63/MGBM Rt. Parietal SB/RT/TMZ 0 26 N N N N N N N N PD <2 5 6 56/M AOA Lt.Temporal SB/RT/TMZ 0 27 N P N P P P P P SD >30 >30 7 37/F AA Rt.Temporal SB/RT/TMZ 0 919 N N N N N P P P SD 25 >33 8 45/F GBM Rt.Frontal SB/RT/TMZ/Mol 0 480 N N N N N  P§  P§ N SD 16 >30 9 43/F AO Rt.Frontal Res/RT/TMZ/SR 0 24 P N N P P N N N PD <2 23 10 71/F GBM Lt.Parietal Resx2/RT/TMZ/ 2 <10 P N N P P N N N SD 5 >27 CE 14 40/M GBM Lt.Front/Temp Res/RT/TMZ 0 551 N N N N N N N N SD 18 >18 Dose 11 54/M GBMRt. Temporal Res/RT/TMZ/ 2 38 Not Tested Not Tested  ND* <2 3 Level 2Mol (3 × 10⁷ 12 35/F AO Lt. Frontal SB/TMZ 0 111 N N N P N  P§ N N SD13 >25 DC/ 13 46/M AA Rt. Parietal Res/RT/TMZ 1 151 Not Tested NotTested  ND* <2 4 dose) 15 51/M GBM Multiple Res/RT/TMZ 0 35 N N N N N NN N SD 4 12 16 33/M AO Rt. Frontal Res/RT/TMZ/Mol 2 985 P N N P P N  P§ P§ SD >14 >14 17 30/F GBM Lt. Parietal Resx2/RT/TMZ/ 1 123 N N N N PNot Tested PD <2 6 CW 18 61/F GBM Bil. Occipital Res/RT/TMZ/BI 1 125 N NN P P N N N PD <2 5 19 63/M GBM Lt. Temporal Res/RT/TMZ/SR 1 199 N N N NN  P§  P§ P SD >13 >13 20 62/M GBM Rt. Temporal Res/RT/TMZ 0 287 N N N NN  P§  P§  P§ PR >13 >13 21 38/F GBM Rt. Hemi Res/RT/TMZ/ 1 27 N N N P NNot Tested PD <2 12 CPT-Bev 22 28/M AA Brain Stem SB/RT/TMZ/Res 0 779 NN N N P N N N SD >12 >12 Abbreviations: M, male; F, female; GBM,glioblastoma multiforme; AA, anaplastic astrocytoma; AO, anaplasticoligodendroglioma; AOA, anaplastic oligoastrocytoma; Temp, temporal; Pa,parietal; Bil, bilateral; Hemi, hemispheric; Res, resection; RT,radiation therapy; TMZ, temozolomide; Mol, molecularly targeted therapy;SB, stereotactic biopsy; SR, stereotactic radiosurgery; CE, carboplatinand etoposide; CW, carmustine-releasing wafer; BI, bevacizumab andirinotecan; No. Prev. Rec, number of previous recurrences; DC IL-12,production of IL-12 p70 by αDC1 (pg/10⁵ cells/24 hours); I, IL- 13Rα2;E, EphA2; Y, YKL-40; G, gp100; Pa, PADRE; P, positive; N; negative; PR,partial response; SD, stable disease; PD; progressive disease; ND*, notdetermined due to early progression before Week 9; TTP, time toprogression.

TABLE 3B ELISPOT (best response) Pt ID I E Y G Pa Dose Level 1 25-49 <2525-49   25-49   100-199 @ 1 (1 × 10⁷ 2 50-99 25-49 25-49   >200   50-99@ DC/dose) 3 <25 <25 <25 25-49 25-49 4 Not Tested 5 <25 25-49 25-49  25-49 25-49 6  100-199 § 100-199 50-99 §  100-199 >200  7  50-99 § <25<25  100-199 § <25 8  50-99 § <25 50-99 §   100-199 § 25-49 9 50-99 <2550-99 @ 100-199 50-99 10 >200    100-199 @ 50-99 @ >200 >200  14 <25  50-99 @ 50-99 @   50-99 @ <25 Dose Level 11 Not Tested 2 (3 × 10⁷ 12 50-99 §  50-99 § 50-99 §  50-99 <25 DC/dose) 13 Not Tested 15 <25 <25<25   100-199 @ 25-49 16 100-199 25-49 <25 >200 >200  17   50-99 @ <2550-99 @ 25-49 50-99 18 25-49 <25 25-49   >200 100-199 19 <25 <25 <25 <25 <25 20 <25 <25 <25 25-49 <25 21 25-49 25-49 50-99 @ 50-99 <25 22 100-199 § <25 25-49     >200 § 100-199 Abbreviations: §, positive onlyafter booster vaccines; @, only a single point, but not two or moreconsecutive points demonstrated 50 or more spots/10⁵ cells (thus notpositive); I, IL-13Rα2; E, EphA2; Y, YKL-40; G, gp100; Pa, PADRE.

7.7.1.2 Clinical Trial Design

This study was designed to assess toxicity and the induction of immuneand preliminary clinical responses of vaccinations with GAA-loaded αDC1and administration of poly-ICLC (Hiltonol®, Oncovir, Inc.). The firstcourse of vaccines consisted of 4 ultrasound-guided intranodal (i.n.)administrations of 1 or 3×10⁷ αDC1/injections every 2 weeks (FIG. 19)rotating each of inguinal and axillary lymph node clusters to minimizethe potential effects of injection-induced trauma in themicroenvironment of the lymph nodes by repeating injections in shortperiods. The first 10 evaluable patients received 1×10⁷ αDC1/injection(Dose Level 1), then 9 received 3×10⁷ αDC1/injection (Dose Level 2). Allpatients received intramuscular (i.m.) injections with poly-ICLC (20μg/kg) twice/week for 8 weeks starting on day 1. Patients exhibitingstable disease or regression of disease without major adverse events(AE) after the 4^(th) vaccination were eligible for additionalvaccinations. Starting at Week 13, these patients were treated with thesame dose of additional vaccinations every 4 weeks to a maximum of 5vaccine injections and i.m. poly-ICLC starting on the day of the firstadditional vaccine and twice/week (1^(st) booster phase). Patients notdemonstrating major AE or tumor progression after the 1^(st) boosterphase were offered the same dose of additional vaccines (every 3 months)and poly-ICLC (every week) for up to three years from the firstvaccination (2^(nd) booster phase).

7.7.1.3 Toxicity Assessment and Stopping Rules

The trial was monitored continuously for treatment-related AE usingNational Cancer Institute Common Toxicity Criteria version 3.0. Thefollowing were considered to be dose-limiting toxicity (DLT) if theywere judged possibly, probably or definitely associated with treatment:≥Grade 2 hypersensitivity; ≥Grade 3 nonhematologic/metabolic toxicity;≥Grade 3 hematological (except for lymphopenia) or metabolic toxicitythat did not subside following 4 weeks temporary cessation of poly-ICLC.Stopping rules were implemented such that a dose level was considered tobe excessively toxic, warranting that accrual be halted, if at any timethe observed rate of DLT was ≥33% and at least 2 DLTs had been observed.

7.7.1.4 Peptides

HLA-A2-restricted peptides used in these studies included: ALPFGFILV(SEQ ID NO:3; IL-13Rα2_(345-353:1A9V)); TLADFDPRV (SEQ ID NO:6;EphA2883-891); IMDQVPFSV (SEQ ID NO:11; GP100_(209-217:M2)); andSIMTYDFHGA (SEQ ID NO:10; YKL-40201-210). αDC1 were also loaded with apan-DR epitope (PADRE), which is a non-natural epitope optimized forhelper T-cell response (see, e.g., Alexander et al, Immunity, 1:751-761,1994). The peptides were synthesized by automated solid-phase peptidesynthesis. Peptides were tested in multiple quality-assurance studiesincluding purity, sterility, identity, potency, pyrogenicity andstability.

7.7.1.5 Vaccine Preparation

For the DC culture, monocytes were obtained from the leukapheresisproduct and purified by the Elutra™ System. The monocytes were culturedin CellGenix antibiotic-free culture medium supplemented with 1000 U/mLGM-CSF and 1000 U/mL IL-4 in sterile cartridges, using the AastromReplicell System. The immature (i)DC were harvested on day 6 andcryopreserved. Before each vaccination, aliquots of frozen iDCs werethawed, further matured and polarized with clinical grade IL-1β (10ng/mL), TNF-α (10 ng/mL), IFN-α (3000 U/ml), IFN-γ (1000 U/mI) andpoly-I:C (20 μg/mI) at 37° C. in 5% CO₂ for 48 hours and loaded with GAApeptides (10 μg/ml) for 4-6 hours. Two hours prior to harvest, the PADREpeptide was added to the cultures. Criteria for release of αDC1included: Sterility by Gram stain and bacteriologic culture; negativeMycoplasma; endotoxin <5.0 E.U./kg of body weight; greater than 70%expression of both CD86 and HLA-DR on αDC1.

7.7.1.6 Collection of PBMCs

Peripheral blood (50-60 ml) was drawn at each visit for vaccine (beforethe vaccine) as well as Weeks 0, 9 and 33. Ficoll-isolated PBMC werecryopreserved in 10% dimethyl sulfoxide/90% FBS.

7.7.1.7 ELISPOT Assays

Enzyme-linked immunosorbent spot (ELISPOT) assays were performed asdescribed previously (see, e.g., Kirkwood et al, Clin. Cancer Res.,15:1443-1451, 2009) with slight modifications. Briefly, batched PBMCsamples were evaluated simultaneously following in vitro stimulationwith autologous, irradiated PBMC loaded with wild-type IL-13Rα345-353,EphA2₈₈₃₋₈₉₁, GP100₂₀₉₋₂₁₇ and YKL-40₂₀₂₋₂₁₁ for a week. A positiveELISPOT response was defined as a 2-fold increase in spot-formingT-cells over pre-vaccine level and at least 10 spots/20,000 cells for atleast two consecutive post-vaccine time points against any antigen.

7.7.1.8 Tetramer Assays

Phycoerythrin (PE)-conjugated HLA-A*0201/ALPFGFILV (SEQ ID NO:3)(IL-13Rα2-tetramer), HLA-A*0201/IMDQVPFSV (SEQ ID NO:11)(gp100-tetramer) and HLA-A*0201/TLADFDPRV (SEQ ID NO:6) (EphA2-tetramer)were produced by the National Institute of Allergy and InfectiousDisease tetramer facility within the Emory University Vaccine Center(Atlanta, Ga.) using the peptide synthesized by the University ofPittsburgh Peptide Production Facility. Fluorescein isothiocyanate(FITC)-conjugated anti-human CD8 was obtained from BD Biosciences. Asingle time-point positive response for a peptide was defined to be(0.1+B) % of all CD8⁺ cells positive by tetramer assay (see, e.g., Webert al, J Immunother., 31:215-23, 2008; and Celis, Cancer, 110:203-14,2007), where B is the percent positive at baseline, which was less than0.01% in all cases. A patient was considered to have responded if he/shehad 2 consecutive single time-point responses for any peptide.

7.7.1.9 Cytokine and Chemokine Assays

Total RNA samples were obtained from PBMC using the PAXgene Blood RNASystem (PreAnalytix, Switzerland). RT-PCR was performed in triplicate,and values were standardized to GAPDH and relative expression of mRNAswas calculated using the ΔΔC_(T) method (see, e.g., Livak andSchmittgen, Methods, 25:402-8, 2001). The Luminex-based assay wasperformed in serum samples as previously described (see, e.g.,Zczepanski et al, Cancer Res., 69:3105-3113, 2009). Pre-tested,multiplex plates (Invitrogen) included standard curves and cytokinestandards (R&D Systems). In situ hybridization with radiolabeled cRNAprobe for CXCL10 was performed as described (see, e.g., Fallert andReinhart, J Virol Methods, 99:23-32, 2002), with autoradiographicexposure times of 14 days.

7.7.1.10 Radiological Response Monitoring

Tumor size was assessed at Weeks 9, 17, 25, and 33, and every 3 monthsthereafter using MRI scans with contrast enhancement. Response wasevaluated by McDonald criteria by gadolinium (Gd)-enhanced T1 weightedimages, area of signal prolongation on T2 weighted images, or acombination of both, based on upon the appearance of the pretreatmentMRI.

7.7.1.11 Other Clinical Endpoints

Overall survival was defined by the interval from study entry to date ofdeath. MRI scans were used to evaluate time to progression (TTP).

7.7.2 Results

7.7.2.1 Summary of Clinical Toxicities

Treatment-related AE are listed for all 22 patients in Table 4. Therewere no grade 3 or 4 toxicities, no deaths on study, and no DLT at anydose through the 1^(st) booster phase. No incidences of autoimmunitywere encountered. Toxicity profiles were comparable across dose levels.Grade 1 or 2 injection site reactions were the most common (82%). Grade1 flu-like symptoms, including fatigue (73%), myalgia (32%), fever(23%), chills/rigors (18%) and headache (32%), were common and usuallylimited to 24 hours after each vaccine. Grade 2 lymphopenia was recordedin one patient (5%).

TABLE 4 Grade 1 Grade 2 Adverse Event No. % No. % Blood/Bone MarrowLeukocytopenia 1 5 Injection site reactions Redness, induration,pruritis, pain 17 77 1 5 Constitutional symptoms Fatigue (lethargy,malaise, asthenia) 16 73 Fever 5 23 Chills/Rigors 4 18 Nausea 7 32Vomiting 1 5 Headache 5 23 2 9 Insomnia 1 5 Light headed/dizziness 2 9Myalgia 7 32 Body ache 6 27 Dermatological Skin rash 3 14 Dry skin 1 5Bruising 2 9 Urticaria 1 5 Pulmonary/Upper Respiratory Rhinitis/Runnynose 1 5 All AE listed were possibly, probably, or definitely related tothe vaccine and/or poly ICLC administration. The numbers represent thenumber of patients (of 22) experiencing a particular event at any pointduring the treatment period, with the highest grade reported for anysingle individual. No grade 3 or grade 4 events observed related totreatment through the 1st booster phase. One patient (Patient 6)demonstrated grade 2 systemic urticaria following the 154th injection ofpoly-ICLC during the 2nd booster phase; this was considered to be DLT.However, because the relationship was unclear and the patient wasprogression-free for 22 months by that time, per IRB approval, thepatient was re-treated with booster vaccines and poly-ICLC followingpre-medication with oral diphenhydramine hydrochloride, and has neverdemonstrated similar reactions again.

7.7.2.2 IL-12 Production by αDC1

As shown in Tables 3A-3B, CD40L-induced IL-12 p70 production levels byαDC1 varied substantially between patients, and positively correlatedwith TTP (p=0.0255; FIG. 10) but not with IFN-γ ELISPOT response,patients' age or tumor types.

7.7.2.3 Induction of Epitope-Specific Immune Responses Against GAAs

All 19 patients who completed the initial course of 4 vaccinations hadPBMCs available for immunological monitoring. Insufficient PBMC wereobtained from Patients 17, 21 and 22 to perform both ELISPOT andtetramer assays; and functional ELISPOT assays were prioritized. Thescheduled first 4 vaccines induced immune reactivity to at least one ofthe vaccine-targeted GAAs in 6 of 10 and 5 of 9 in Dose Levels 1 and 2,respectively, by either IFN-γ ELISPOT or tetramer assays (Tables 3A-3B).In patients 6, 7, 8, 16, 19, 20 and 22, some readouts reached thecriteria for positive response following booster vaccines (indicated by§ in Table 3B). In summary, 11 of 19 (58%) evaluable patients showedpositive response after the initial 4 vaccinations, and 3 of 19(Patients 8, 19 and 20; 16%) showed positive response only after boostervaccines.

Positive response rates (either by tetramer or ELISPOT) did not showsignificant differences across the two αDC1 doses per Fisher's exacttest. Furthermore, the magnitudes of ELISPOT response, based on thesummation of positive spots from Week 3 through 9, were comparableacross the two αDC1 dose levels (Wilcoxon test). Therefore, the timecourse of IFN-γ ELISPOT responses is presented by combining results fromboth dose levels (FIG. 11). The gp100 epitope demonstrated the highestmagnitude of response among the GAA peptides tested (p=0.0001, 0.0003and 0.0005 against IL-13Rα2-, EphA2- and YKL-40-derived peptides,respectively, by Wilcoxon test). For the other epitopes, boostervaccines appeared to improve the induction of specific responses. Atemporary decline of responses was typically observed at Week 13, whichmay reflect that some patients who demonstrated positive responses byWeek 9 did not participate in the booster phase due to tumor progression(Patients 2, 9, 18 and 21) or lymphopenia (Patient 10), resulting inoverall reduction of response when data are pooled for all patients.Patient 10 demonstrated the highest magnitude of IFN-γ ELISPOT responsesagainst IL-13Rα2- and gp100-derived epitopes as well as PADRE (FIG. 12)but tetramer analyses on this patient yielded no responses (Table 3A).Patient 6, who demonstrates stable disease for longer than 30 months,developed durable and high level responses in tetramer (FIG. 13) andELISPOT assays.

7.7.2.4 Induction of Type-1 Cytokine and Chemokine Responses

RT-PCR analyses of PBMC (FIGS. 14 and 15) revealed up-regulation of mRNAexpression for several type-1 cytokines and chemokines, specificallyIFN-α1, CXCL10 and TLR3, at both post-1^(st) vaccine and post-4^(th)vaccine. IFN-γ was found to be up-regulated after the 4^(th) vaccine,but not after the 1^(st) vaccine, suggesting that the IFN-γup-regulation may be associated with the induction of adaptive, ratherthan innate, immune response. CCL22, which is known to attractregulatory T-cells (see, e.g., Muthuswamy et al., Cancer Res.68:5972-5978, 2008), and CCL5 levels decreased in paired-analyses ofpost-1^(st) vaccine samples. Perforin, Granzyme B, COX-2 and Foxp3levels did not change significantly.

A panel of cytokines and chemokines was evaluated at protein levels inavailable pre-vaccine and post-vaccine serum samples from 5 patients(FIG. 16). Among them, IFN-α, CXCL10, IL-15, MCP-1 and MIP-1β weresignificantly up-regulated in post-vaccine sera. IL-17 was underdetectable ranges in both RT-PCR and serum analyses.

In addition, three of five available tumors resected due to post-vaccineradiographic progression expressed mRNA for CXCL10, which is a criticalchemokine for effective trafficking of CD8⁺ T-cells to brain tumor sites(see, e.g., Nishimura et al., Cancer Res 66:4478-4487, 2006; and Fujitaet al., Cancer Res 69:1587-1595, 2009) (FIG. 17 for a representativecase). These data suggest that the current regimen induces systemic,poly-functional immune responses in generally immunosuppressed patientswith malignant glioma.

7.7.2.5 Immunohistochemistry Data

Immunohistochemistry data for 7 cases GAA cases are summarized in Table5. These data suggest that expression of gp100 may be very low inprimary high grade gliomas. For immunohistocemistry, the followingpolyclonal antibodies (Ab) and corresponding secondary Ab were used:anti-human(h)IL-13Rα2 (goat IgG; R&D Systems); anti-human EphA2 (H-77)(rabbit IgG; Santa Cruz Biotechnology); anti-human YKL-40 (rabbit IgG;Quidel) and anti-human gp100 (goat IgG; Santa Cruz Biotechnology).

TABLE 5 Pre vs. Post Case # vaccine IL-13Rα2 EphA2 YKL-40 gp100 1 (GBM)Pre 2*  3* 2 0* Post 2*  1* 2 0* 2 (GBM) Pre 1*  2* 1 0* 5 (GBM) Post 1 2 2 1  9 (AO) Pre 1* 2 1 0* Post 1* 1 1 0* 10 (GBM) Post 2* 1 2 0* 12(AO) Pre 2  2 1 1* 14 (GBM) Pre 3  2 2 0  Expression of each GAA wasgraded as follows: grade 0, negative; 1, weakly positive; 2, moderatelypositive; 3, strongly positive. Numbers with asterisks indicate that thepatient demonstrated positive ELISPOT or tetramer response against theantigen. Pre-vaccine does not mean that tumor tissues were obtainedimmediately before the vaccination, but obtained at variable time pointsbefore the vaccine, including the initial diagnostic biopsy orresection. Likewise, post-vaccine tissues were obtained at variable timepoints following the last vaccine because re-resection was not alwaysindicated.

7.7.2.6 Clinical Outcomes

Two patients (Patient 1 and 20) experienced objective clinical tumorregressions (response rate=9%). Both patients were non-responders byELISPOT, but tetramer responders. Patient 20 with recurrent GBMdemonstrated complete response based on disappearance of the Gd-enhancedmass at Week 17 post-vaccine compared with the baseline MRI, which wasdurable and ongoing for at least 13 months since the initiation of thetreatment (FIG. 18A-I). Patient 1 with recurrent GBM exhibited a partialresponse at Week 9. Following two booster vaccines, the Gd-enhancedlesion enlarged. Biopsy of the lesion, however, revealed intensiveinfiltration of CD8⁺ T-cells and CD68⁺ macrophages and no evidence ofmitotically active tumor (FIG. 18J-L). Then, this patient received oneadditional vaccine before the recurrence at 7 months after the initialvaccine. Nine patients (41%; 4 and 5 with GBM and AG, respectively) wereprogression-free for at least 12 months. Five patients remainedprogression-free (Table 3A) and continued to receive booster vaccines.Median TTP are 4 and 13 months for GBM and AG, respectively (FIG. 20).

7.7.3 Conclusion

The study described in this Example evaluated αDC1-based vaccines loadedwith novel GAA-derived peptides, in combination with poly-ICLC. Thefindings demonstrate safety and immunogenicity as well as preliminaryefficacy of the approach.

7.8 Example 8

This example describes a study of the safety and efficacy of atherapeutic regimen for adults with recurrent WHO grade II gliomas thatcomprises vaccination with HLA-A2-restricted glioma antigen-peptides incombination with poly-ICLC.

7.8.1 Rationale

This Example describes a vaccination regime that is designed toefficiently induce anti-tumor T-cell responses in patients withrecurrent WHO grade II glioma. The regime combines subcutaneousinjections of glioma-associated antigen (GAA)-derived cytotoxicT-lymphocyte (CTL) epitope-peptides with simultaneous intramuscular(i.m.) administration of poly-ICLC.

Adults with supratentorial low-grade glioma (LGG) have a significantrisk (24%) of tumor progression 2 years following treatment with surgeryor surgery followed by radiation therapy (RT). The study described inthis Example has both immunoprophylactic and immunotherapeutic potentialto reduce the risk of tumor recurrence, which may translate intoimproved survival. Therapeutically, the immunotherapy approach maysuppress the expansion of indolently growing neoplastic low grade IItumor cells. Prophylactically, the approach may prevent anaplastictransformation, which occurs in about one-half of recurrent LGG. Theslower growth rate of LGG (in contrast to malignant gliomas) shouldallow sufficient time to repeat multiple immunizations, which may leadto the induction of high levels of GAA-specific immunity. In addition,poly-ICLC has been demonstrated to enhance the vaccine effects inpreclinical brain tumor models (see, e.g., Zhu et al., J. Transl. Med.,5: 10, 2007), and to be safe in malignant glioma patients (see, e.g.,Salazar et al., Neurosurgery, 38: 1096-1103, 1996). Therefore, wehypothesize that this form of vaccine in combination with poly-ICLCtreatment will induce potent anti-glioma immune response, and will besafe.

7.8.2 Objectives

This Example describes a vaccine study in adults with recurrent WHOgrade II glioma. The objectives of this Example include collection ofimmunological and safety data that may be used in additional studies.The patients in the study described in this Example may be followed fora minimum of 2 years, so that the actual 2-year overall survival (OS),6-month and 2-year progression-free survival (PFS) rates may bedetermined in an exploratory manner.

7.8.2.1 Induction of GAA-Specific T-Cell Response

The response rate and magnitude of immune response in post-vaccineperipheral blood mononuclear cells (PBMC) against the GAA-peptides inresponse to this form of vaccine may be determined usingIFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

7.8.2.2 Safety

The incidence and severity of adverse events associated with the vaccineregime may be assessed, with an early stopping rule based on thefrequency of Regimen Limiting Toxicity (RLT).

7.8.2.3 Clinical Response

Radiological response may be determined using the standard WHO responsecriteria. 6-month and 2-year progression-free survival (PFS) may beevaluated in an exploratory manner, based on serial magnetic resonanceimaging (MRI) scans.

7.8.2.4 Tumor Tissues for Biological Correlates

For patients who develop progression, biopsy/resection may beencouraged. Whenever post-vaccine tumor tissues are available, they maybe analyzed for GAA expression status and infiltration of GAA-specificT-cells.

7.8.3 Patient Selection

7.8.3.1 Eligibility Criteria

Pathological criteria—Patients have recurrent supratentorial WHO gradeII astrocytoma, oligoastrocytoma or oligodendroglioma that ishistologically confirmed either by the previous biopsy or resection, orat the time of re-operation (re-operation before entry to the currentstudy is allowed; however post-surgery Decadron must be off for at least4 weeks before administration of the first vaccine). Patients in thisstudy should have received prior external beam radiotherapy and/orchemotherapy. With regard to the prior therapy, patients in this studyshould have had treatment for no more than 2 prior relapses. Relapse isdefined as progression following initial therapy (i.e. radiation+/−chemoif that was used as initial therapy). The intent therefore is thatpatients in this study should have had 3 prior therapies (initialtherapy and treatment for 2 relapses). If the patient had a surgicalresection for relapsed disease, and no anticancer therapy was institutedfor up to 12 weeks, and the patient undergoes another surgicalresection, this is considered as 1 relapse.

Patients in this study should be HLA-A2 positive based on flowcytometry.

Patients in this study should have recovered from the toxic effects ofprior therapy: 4 weeks from any investigational agent, 4 weeks fromprior cytotoxic therapy and/or at least two weeks from vincristine, 4weeks from nitrosoureas, 3 weeks from procarbazine administration, and 1week for non-cytotoxic agents, e.g., interferon, tamoxifen, thalidomide,cis-retinoic acid, etc. (radiosensitizer does not count). With regard toprevious radiation therapy (RT), there must be at least 6 months fromthe completion of RT (or radiosurgery).

Patients in this study should be >18 years old.

Patients in this study should have a Karnofsky performance status of >60(Appendix I).

Female patients in this study of child-bearing age should havedocumented negative serum βHCG.

Patients in this study should be free of systemic infection. Patientswith active infections (whether or not they require antibiotic therapy)may be eligible after complete resolution of the infection. Patients onantibiotic therapy should be off antibiotics for at least 7 days beforebeginning treatment.

Patients in this study should have adequate organ function as measuredby white blood count ≥2500/mm³; lymphocytes ≥400/mm³; platelets≥100,000/mm³, hemoglobin ≥10.0 g/dL, AST, ALT, GGT, LDH, alkalinephosphatase within 2.5× upper normal limit, and total bilirubin ≤2.0mg/dL, and serum creatinine within 1.5× upper limit of normal limit.Patients in this study should have coagulation tests and PT and PTTshould be within normal limits.

7.8.3.2 Exclusion Criteria

Patients in this study should be excluded if they have presence ofgliomatosis cerebri, cranial or spinal leptomeningeal metastaticdisease.

Even if the initial diagnosis was WHO grade II glioma, if thepathological diagnosis for the recurrent disease demonstratetransformation to higher grade (i.e. WHO grade III or IV) gliomas,patients should be excluded from this study.

Patients in this study should be excluded if they are undergoingconcurrent treatment or medications including: radiation therapy;chemotherapy; interferon (e.g. Intron-A®); allergy desensitizationinjections; growth factors (e.g. Procrit®, Aranesp®, Neulasta®);interleukins (e.g. Proleukin®); and/or any investigational therapeuticmedication.

Patients in this study should not have had prior autoimmune disordersrequiring cytotoxic or immunosuppressive therapy, or autoimmunedisorders with visceral involvement. Patients in this study with anactive autoimmune disorder requiring these therapies also should beexcluded. Mild arthritis requiring NSAID medications should not beexclusionary.

Patients in this study should be excluded if they have usedimmunosuppressives within four weeks prior to entering the study or ifthey anticipate use of immunosuppressive agents. Dexamethasone, or othercorticosteroid medications, if used peri-operative period and/or duringradiotherapy, should be tapered by patients and discontinued at leastfour weeks before administration of the first vaccine in the study.Topical corticosteroids and Inhaled steroids (e.g.: Advair®, Flovent®,Azmacort®) should be acceptable.

Patients in this study should be excluded if they have another cancerdiagnosis, except that the following diagnoses may be allowed: squamouscell cancer of the skin without known metastasis; basal cell cancer ofthe skin without known metastasis; carcinoma in situ of the breast (DCISor LCIS); carcinoma in situ of the cervix; and/or any cancer withoutdistant metastasis that has been treated successfully, without evidenceof recurrence or metastasis for over 5 years.

Patients in this study should be excluded if they have known addictionto alcohol or illicit drugs.

Because patients with immune deficiency are not expected to respond tothis therapy, HIV-positive patients should be excluded from the study.

7.8.4 Peptide Vaccine

7.8.4.1 Peptides

The following peptides may be included in the vaccine formulation:IL-13Rα2₃₄₅₋₃₅₃ 1A9V (ALPFGFILV; SEQ ID NO:3); EphA2₈₈₃₋₈₉₁ (TLADFDPRV;SEQ ID NO:6); Survivin96-104:M2 (LMLGEFLKL; SEQ ID NO:7); WT1₁₂₆₋₁₃₄:Y1(YMFPNAPYL; SEQ ID NO:8); and Tetanus Toxoid (TetA830)(AQYIKANSKFIGITEL; SEQ ID NO:9).

All peptides may be synthesized and the synthetic peptides may bepurified by HPLC. The identity of the synthetic peptides may beconfirmed by verifying their mass and amino acid sequences by massspectrometry. Each lot of peptide may be evaluated as required by theFDA for identity, purity, sterility and pyrogenicity.

The peptides may be vialed under GMP conditions and saved at −70° C.Stability of lyophilized peptides may be tested annually by massspectroscopy.

7.8.4.2 Other Agents

Montanide ISA-51 (SEPPIC Inc., Fairfield, N.J.) may be used as anadditional agent in the peptide vaccines.

7.8.4.3 Dosage and Preparation

An aqueous solution (500 μL) containing each of four HLA-A2-restrictedGAA peptides (300 μg/peptide) and the tetanus peptide (Peptide-tet; 200μg) may be mixed 1/1 with Montanide ISA-51 to form one water-in-oilemulsion (i.e. the total volume/injection is 1 mL).

7.8.4.4 Administration

Patients in this study may be vaccinated subcutaneously in the right orleft upper arms with intact draining axillary nodes. In case patients donot possess intact axillary lymph nodes as the draining nodes, thevaccines may be administered in the upper thigh on the same side withintact inguinal lymph nodes.

The vaccine may be administered on weeks on Weeks 0, 3, 6, 9, 12, 15, 18and 21.

7.8.5 Poly-ICLC

Poly-ICLC may be prepared and packaged in the GMP facility of Bioserv,Corporation (San Diego, Calif.). Poly-ICLC may be supplied in vialscontaining 1 cc of translucent solution with a concentration of 2 mg percc. Poly-ICLC is stable at room temperature for several days, but may bestored refrigerated at about 40° F.

7.8.5.1 Dosage and Administration

Poly-ICLC may be administered intramuscularly at doses of 20 μg/kg andup to 1640 μg/injection, with two injections on days 0 and 4 followingeach vaccination.

The first course of poly-ICLC administration (20 μg/kg i.m. and up to1640 μg/injection) may be administered on the day of the firstGAA/TT-vaccine and on day 4 after the vaccine. For each of the followingrepeated vaccinations (on Weeks 3, 6, 9, 12, 15, 18 and 21), poly-ICLC(20 μg/kg i.m. and up to 1640 μg/injection) may be administered on theday of the vaccine and on day 4 after the vaccine.

With regard to the injection sites, as poly-ICLC is expected to enhancethe antigen-presentation process in the draining lymph nodes, poly-ICLCshould be administered i.m. within the close vicinity to the previouspeptide-injection site (e.g., less than 3 cm from the center of theprevious peptide injection sites).

Poly-ICLC should be administered intramuscularly (i.m.) using steriletechnique, as supplied from the vial, and in the amount prescribed forthe patient's weight (up to 1640 μg/injection). Vital signs may bemonitored before and for at least 20 minutes after the first treatment.

7.8.6 Treatment Plan

The study described in this Example may employ two cohorts of patientsto assess the immunogenicity, safety and clinical efficacy of theGAA/TT-peptide vaccine and poly-ICLC in HLA-A2+ patients with recurrentWHO grade II gliomas. Because the peptide vaccine is sequesteredlocally, and the immune response occurs primarily locally and in thedraining lymph nodes, the dose of the vaccine should not need to bescaled up proportionately to the size (by weight or body surface area)of the recipient, as might be done for a drug whose effect is related toits distribution in body fluid. With regard to the dose of poly-ICLC, afixed dose (20 μg/kg/injection and up to 1640 μg/injection) may beemployed, which has been demonstrated to be safe and to inducebiological responses in patients with malignant glioma (see, e.g.,Salazar et al., Neurosurgery, 38: 1096-1103, 1996).

7.8.6.1 Schedule

Patients may be treated with subcutaneous injections of GAA/TT-vaccineson Weeks 0, 3, 6, 9, 12, 15, 18 and 21. I.m. poly-ICLC may beadministered (20 μg/kg i.m. and up to 1640 μg) on the day of, and on day4 after each vaccine (e.g. if the vaccine is administered on Thursday,poly-ICLC may be administered on the day of vaccine and the followingMonday). Each vaccine may be administered within 2 hours before or afterthe i.m. poly-ICLC administration.

Patients may be evaluated for any possible adverse event, regimenlimiting toxicity (RLT) as well as clinical/radiological responses byclinical visits and MRI scanning. MRI scans may be performed on Weeks 0,12 and 24. If the scan on Week 12 demonstrates unequivocal tumorprogression, the patient may be withdrawn.

Peripheral blood mononuclear cells (PBMC) obtained before the initialvaccine may be used as the base-line sample. If patients demonstrate anypositive response in the two immunological assays (ELISPOT or Tetramerwithout RLT or tumor-progression, these patients may be offeredadditional GAA/TT-vaccines (see, e.g., Section 7.8.6.2) starting anytime between on Week 34-40, and every 3 months thereafter until patientsdemonstrate tumor-progression, loss of immune response or RLT.

7.8.6.2 Additional Therapy

On Weeks 0 (baseline), 12, 15, 18, 21, and 24, patients' PBMC may beevaluated for the presence of GAA-specific T-cell responses against GAApeptides. If such a response is observed for any of the GAA peptides,the patient may undergo additional vaccinations with the GAA(s) thatdemonstrated the persisting response as well as poly-ICLC starting anytime between on Week 34-40, and every 12 weeks thereafter up to 2 yearsfrom the initial vaccination. Additional PBMC samples may be obtainedevery 12 weeks (at the same visits for vaccine administrations) forimmunological monitoring. Additional vaccines may be terminated in anyof the following conditions: 1) tumor progression; 2) RLT; or 3)negative immunological response in two consecutive time points.

7.8.6.3 Dose Modification

7.8.6.3.1 Dose Modification for Poly-ICLC

For any Grade 2 or greater flu-like symptoms, including fever andfatigue, poly-ICLC may be discontinued until symptoms return to Grade 0.If Grade 2 or greater flu-like symptoms occur on the day of avaccination, and if the symptoms do not return to Grade 0 by day 4 afterthe vaccination, the next poly-ICLC administration on day 4 after thevaccination may be skipped. If the patient is symptom-free on day 4(Grade 0), poly-ICLC may be resumed at 50% of the original dose. IfGrade 2 or greater flu-like symptoms occur following the poly-ICLCadministration on day 4 after a vaccination, in the next vaccine cycle,two poly-ICLC administrations (on day 0 and 4 following the vaccines)may be given at 50% of the original dose. Pretreatment withacetaminophen 650-1000 mg or with any NSAID may be given. If furtherdosing is well tolerated, the original dose may be subsequentlyre-instituted.

In the case of hepatic enzyme elevation >4× baseline, or any otherunforeseen intolerable side effects of grade 2 or greater, poly-ICLC maybe discontinued until that toxicity has reduced to Grade 1 or less.Poly-ICLC may then be re-administered at one-half the original dose, andthe patient may be closely observed. If the poly-ICLC cannot bere-initiated in the next vaccine cycle, the patient may be withdrawn forthe RLT.

For patients who demonstrated grade 3 or lesser degree of lymphopenia attheir study entry (our eligibility criterion requires 400 cells/μL),occurrence or continued presence of grade 3 lymphopenia during the studydoes not mandate the discontinuation of poly-ICLC. However, poly-ICLCmay be suspended in case of grade 4 lymphopenia. Also, if theattribution of poly-ICLC is strongly suspected even for grade 3lymphopenia, poly-ICLC administrations may be suspended. In these cases,re-administration at one-half the original dose may be allowed whenabsolute lymphocyte counts come back to at least 400/μL.

Patients may remain on the original dose for grade 1 toxicities.However, the dose may be reduced to 50% for grade 2 hematologic ornon-hematologic toxicity (except for transient fever and fatigue asoutlined earlier in this section). If at the 50% dose level there is notoxicity for a minimum of 2 weeks, the dose may be escalated back to thestarting dose. Subsequent toxicities, should they occur, may require adose reduction to 50%, and no further escalations may be allowed. Iftoxicity reoccurs at the reduced dose, the patient may be taken offtreatment.

7.8.6.3.2 Dosing Delay for the Peptide Vaccines

In circumstances where poly-ICLC administration is suspended, if theevent is not attributable to the peptides/ISA-51 vaccine, vaccineadministrations may not be suspended. If the event is attributable toboth poly-ICLC and peptide-vaccines, both may be suspended. If anadverse event is considered to be solely due to the peptides/ISA-51vaccines, but not poly-ICLC, the vaccine and poly-ICLC administrationsmay be suspended. In circumstances where assessment of an adverse eventis limited, such as by intercurrent illness, or when laboratory studiesare required to assess for other causes of toxicity, the vaccineschedule may be interrupted for up to 4 weeks. Delay of one vaccineadministration by up to 4 weeks will not be considered a protocolviolation if due to an adverse event, regardless of attribution. If oneor more vaccines is delayed by 4 weeks due to an adverse event,regardless of attribution, treatment should be discontinued.

Patients may be observed for regimen limiting toxicity (RLT) throughoutthe study. The following are considered to be RLTs if they are judgedpossibly, probably or definitely associated with treatment. Should theyoccur, individual patients may be taken off study and no furtherinjections may be given.

≥Grade 2 or more bronchospasm or generalized uticaria(hypersensitivity).

≥Grade 2 or more allergic reaction, such as exfoliative erythroderma,anaphylaxis, or vascular collapse.

≥Grade 2 or more autoimmune disease (e.g. hypothyroidism, autoimmuneencephalitis).

Any ≥Grade 3 toxicity possibly, probably, or definitely related to thevaccine with particular attention to the following events.

≥Grade 3 injection site reaction due to peptide-vaccine or poly-ICLCadministration.

≥Grade 3 hematological or hepatic toxicity.

≥Grade 3 neurotoxicity: signs and symptoms that may indicate eithertumor progression or an inflammatory immune response (i.e., pseudo-tumorprogression) that requires a biopsy or resection with pathologicfindings of inflammatory/lymphocytic infiltration.

≥Grade 3 nausea and vomiting without sufficient antiemetic prophylaxisare not considered as RLTs.

Dosing delays >4 weeks for either poly-ICLC or peptide vaccines.

Therapy may be discontinued for the following reasons: (i) Regimenlimiting toxicity—as defined above; (ii) disease progression—at least a20% increase in the sum of the longest diameter of target lesion or theappearance of contrast enhancement in a previously non-enhancing tumor.However, if pseudo-tumor progression is suspected, then the patient maybe placed on dexamethasone, up to 4 mg/day, and reimaged 4-8 weekslater. If they require >4 mg/day dexamethasone, or if their repeatimaging study continues to meet the criteria for disease progression,the patient may be taken off study and further study treatment may bediscontinued. However, if their steroid dose is <4 mg/day and if theirrepeat imaging does not meet the criteria for disease progression, thenthe patient may continue in the study and receive study treatment asprescribed herein. Any cases of suspected tumor progression orpseudo-tumor progression should be reviewed to determine whether thesubject should remain in the study. (iii) Intercurrent illness thatprevents further administration of the vaccine or poly-ICLCadministration. (iv) Pregnancy: Pregnant patients will continue to befollowed for the duration of the pregnancy.

7.8.6.4 Treatment Duration

In the absence of treatment delays due to adverse event(s), treatmentmay continue for 21 weeks (8 vaccinations) or until one of the followingcriteria applies: Regimen Limiting Toxicity (RLT); disease progression;and/or intercurrent illness that prevents further administration oftreatment.

7.8.6.5 Concomitant Treatment

7.8.6.5.1 Acceptable

For fever, acetaminophen may be utilized (325 mg tabs, 1 or 2 p.o. every4 hours). Pre-treatment of patients with acetaminophen may be institutedas warranted by side effects of poly-ICLC. Fevers lasting more than 8hours after treatment may be evaluated in terms of potential infection.

For mild local pain, oral opiates may be planned (oxycodone, 5-10 mgp.o. every 3-4 hours). Pain that is of more than mild-moderate grade maybe investigated for sources other than the therapy, and managedaccordingly.

Dexamethasone (or similar corticosteroid medications) should not be usedfor at least 4 weeks prior the initiation of the vaccine/poly-ICLCtherapy (Week 0). Dexamethasone (up to 4 mg/day) may be used in thesetting of pseudo-tumor progression, and tapered/discontinued as soon aspossible.

Anti-seizure medications should be used as indicated.

Antiemetics, if necessary, may be administered.

Other acceptable medications may include: Topical corticosteroids;nonsteroidal anti-inflammatory agents; anti-histamines (e.g. Claritin®,Allegra®); chronic medications except those listed in Section 7.8.6.5.2;Influenza vaccines (these should be administered at least two weeksprior to the initiation of the study vaccines or at least two weeksafter the 8^(th) (last) vaccine); and/or corticosteroid medicationsadministered parenterally or by inhalation (e.g.: Advair®, Flovent®,Azmacort®).

7.8.6.5.2 Unacceptable

Unacceptable medications may include interferon therapy (e.g.Intron-A®); chemotherapy; allergy desensitization injections; growthfactors (e.g. Procrit®, Aranesp®, Neulasta®); interleukins (e.g.Proleukin®); other investigational medications; and/or illicit drugs.

7.8.7 Correlative/Special Studies

7.8.7.1 Immunological Monitoring

7.8.7.1.1 Enzyme Linked Immuno-SPOT (ELISPOT) Assays

Frequencies of glioma associated antigens (GAA)-responsive T-lymphocyteprecursors in peripheral blood mononuclear cells (PBMC) prior to andafter, administration of the GAA-peptide based vaccine may be measuredby ELISPOT assay. The biological responses measured by ELISPOT may bedone at the same time point at least for one individual patient to avoidinter-assay variability. Successful vaccination stimulates clonalpopulations of T cells that are capable of secreting cytokines in anantigen-specific, MHC-restricted fashion. The ELISPOT assay may beutilized to evaluate GAA-specific immune responses of CD8+ T-cellpopulations as well as CD4+ T cells that react against the helper TTpeptide. IFN-γ production may then be evaluated to assess Type-1 T-cellresponse.

A subject may be considered to have responded, if at any of twoconsecutive post-vaccine time points against the same antigen[s] (Weeks12, 15, 18, 21 and 24), the number of spots is double that at baseline,and there are at least 10 spots/20,000 cells, and if the number of thepost-vaccine spots is at least three times the standard-deviation of thepre-vaccine value. Response can be to any one antigen.

7.8.7.1.2 Tetramer Analysis of GAA-Reactive T Cells in Patient's PBMC

Tetramer analyses allow for evaluation of the presence of GAA-specificCD8⁺ T-cells in peripheral blood with a great sensitivity without invitro re-stimulation of the cells. It is expected, based on previousdata available from patients with malignant glioma, that significant (alog or more) increase in the frequency of peptide-responsive CD8⁺ Tcells may be observed in some, but not all, patients immunized withtumor-antigen based vaccines. In an exploratory manner, these PBMCs maybe also evaluated for surface expression of an integrin receptor verylate antigen (VLA)-4, which has been implicated to confer T-cell homingto CNS tumors (see, e.g., Zhu et al., J. Transl. Med., 5: 10, 2007) andchemokine receptors (e.g. CXCR3 and CCR5). Procedures for tetrameranalysis are well established.

Tetramer assays may be done at baseline and at 5 time points aftervaccinations (Weeks 12, 15, 18, 21 and 24). A single time-point positiveresponse for a peptide to be (1+B) % of all CD8⁺ cells positive bytetramer assay may be defined, where B is the percent positive atbaseline, which is usually less than 0.1%. In analogy to the definitionof ELISPOT response, a patient may be considered to have responded ifhe/she has two consecutive single time-point responses for any peptide.

7.8.7.1.3 Flow Cytometric Analyses of Lymphocyte Subsets

Numbers of CD4+ and CD8+ T cells as well as CD4+/Foxp3+ T regulatorycells at serial time points pre- and post-vaccines may be evaluated.

7.8.7.1.4 Analyses of Autoimmunity in Sera

Banked sera may be evaluated for presence of auto-antibodies.

7.8.7.2 Evaluation of Primary and Recurrent Tumor Tissues

GAA-expression in the patients' available tumor tissues may be evaluated(either pre-vaccine or after progression post-vaccines; or both) byimmunohistochemistry (IHC) and reverse transcriptase-polymerase chainreaction (RT-PCR).

If tumors recur following vaccinations, it may be critical to evaluatehow tumors escape the effects of vaccines. To this end, the followingspecific issues may be evaluated as much as the tissue-availabilityallows: (i) Antigen-loss: IHC and RT-PCR may be used to assess whetherthe recurrent tumors express the targeted GAAs, HLA-A2, and antigenprocessing machinery components, such as transporter associated withantigen processing; (ii) up-regulation of anti-apoptotic molecules:although Survivin may be targeted, other anti-apoptotic molecules may beup-regulated, e.g., cFLIP (cellular FLICE (Fas-associated deathdomain-like IL-1ß-converting enzyme) inhibitory protein); and (iii)immune cell infiltration: one reason tumors may escape a vaccine-inducedimmune response is through the failure of reactive T cells to infiltratethe tumor. To examine this, whenever freshly resected tumor tissues (notfixed or frozen) are available, tumor infiltrating lymphocytes (TILs)may be isolated and their numbers, phenotype, and antigen-specificitymay be characterized using HLA-A2 tetramers for each of GAAs. Usingmulti-color flow-cytometry, the function and viability of tetramer⁺ TILsmay be determined by staining for perforin/IFN-γ and Annexin-V,respectively. Control tissues may include pre-vaccine tumors (ifavailable) and recurrent tumors from patients not in the study. Thesestudies may allow for evaluation of whether vaccine-induced T-cellsefficiently traffic to the brain tumor site and maintain their functionand viability.

7.8.8 Study Parameters

This study may be conducted on an outpatient basis, with patientsscheduled to be evaluated on weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24.After this period, if patients do not receive additional vaccines,patients may be off study, and they may be clinically followed every 2-4months thereafter as usually done for patients with the same tumor type.If patients are found to have progressing tumors, other therapies, suchas chemotherapy or resection, may be offered. If patients receiveadditional vaccines, such additional vaccines may be administered every12 weeks, and clinical, immunological and radiological (MRI) monitoringmay be performed at every visit (q12 weeks) until the patients withdraw.Subjects with a complete response may be retreated with additional twovaccinations, with 12-week intervals, and then followed. Vaccinationsmay be halted for any patients with progressive disease or unacceptabletoxicity at any time during the scheduled vaccinations.

7.8.8.1 Pre-Treatment (Screening and Baseline Data)

The following procedures may be undertaken before treatment proceeds:informed Consent should be obtained before initiation of screening; HLAtyping (flow-cytometric evaluation for HLA-A2 positivity); documentationof diagnosis (pathological); complete history and physical examination(with vital signs and weight), including neurological examinations andperformance status; vaccine sites are to be designated with confirmationof intact draining lymph nodes; demographic information should berecorded; CBC and platelets with differential should be evaluated;PT/PTT should be evaluated; Chemistry should be evaluated, includingelectrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, Alkphos, total bilirubin, LDH, calcium and albumin; GGT, phosphorus, andmagnesium should be evaluated; Blood for in vitro assays should betaken; HGBA1C for patients with diabetes mellitus should be performed;ECG and echo cardiogram should be performed for patients with cardiacsymptoms, history or current disease; urinalysis should be performed;MRI of the brain to evaluate the baseline status of disease should beperformed; and/or women of child-bearing potential should beadministered a serum beta-HCG pregnancy test.

7.8.8.2 Evaluation During Treatment

The following procedures may be undertaken as treatment proceeds.Pre-Administration (Weeks 0, 3, 6, 9, 12, 15, 18 and 21 before vaccineadministration on the day of vaccination): history and physicalincluding vital signs, weight, Karnofsky performance status andneurological function; blood for in vitro assays should be taken;Chemistry should be evaluated, including electrolytes, creatinine, bloodurea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin (Except for Week 0); AED levels should be evaluatedif clinically indicated; patients should be screened for adverse eventsfrom previous doses, to include neurological evaluation and skinexamination (injection sites); and/or Mill should be performed (only onWeek 12 among these vaccine injection visits).

Following vaccine administration, all patients should be closelyobserved for adverse events for at least 20 minutes following eachadministration of GAA-peptide vaccine. On the same day, poly-ICLC (i.m.20 mg/kg) may be administered within 2 hours before or after thevaccine, and monitored at least 20 minutes after the poly-ICLCinjection.

7.8.8.3 Week 24 (Post 8 Vaccinations) Evaluation

After the vaccination cycle is complete, the following procedures may beundertaken: history and physical including vital signs, weight,Karnofsky performance status and neurological function; Blood for invitro assays should be taken (Except for Week 3, 6 and 9); CBC andplatelets with differential should be evaluated (Except for Week 0);Chemistry should be evaluated, including electrolytes, creatinine, bloodurea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin (Except for Week 0); AED levels should be evaluatedif clinically indicated; patients should be screened for adverse eventsfrom previous doses, to include neurological evaluation and skinexamination (injection sites); and/or Mill should be performed.

7.8.8.4 Evaluation with Additional Vaccines (Cases with AdditionalVaccines)

Prior to administration with additional vaccines, the followingprocedures may be undertaken: history and physical including vitalsigns, weight, Karnofsky performance status and neurological function;Blood for in vitro assays should be taken (Except for Week 3, 6 and 9);CBC and platelets with differential should be evaluated (Except for Week0); Chemistry should be evaluated, including electrolytes, creatinine,blood urea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin (Except for Week 0); AED levels should be evaluatedif clinically indicated; patients should be screened for adverse eventsfrom previous doses, to include neurological evaluation and skinexamination (injection sites); and/or MRI should be performed.

Following administration with additional vaccines, all patients shouldbe closely observed for adverse events for at least 20 minutes followingeach vaccination. Additional vaccines may be terminated in any of thefollowing conditions: 1) tumor progression; 2) RLT; or 3) negativeimmunological response in two consecutive time points after initiationof additional vaccines.

TABLE 6 Study Calendar Pre-Vaccination every 12 Consent/ Wks forManagement Table HLA-typing/ Within Treatment (Week) additional Studies& Tests path 4 Wks 0 3 6 9 12 15 18 21 24 vaccines * Informed consentfor HLA-typing X HLA-typing X Informed consent for treatmentX-------------------X (if HLA-A2 is positive) Pathology review XVaccination^(#) X X X X X X X X X CBC and Platelets with differential XX X X X X X X X X Coagulation tests (PT and PTT) X Chemistry^(##) X X XX X X X X X X GGT, Phosphorus, Magnesium X AED if clinically indicated XX X X X X X X X Demographics X Concurrent Medications X←------------------------------------------→ Urinalysis X β-HCG (womenof childbearing X potential) EKG or Echocardiogram^(###) X (ifclinically indicated) Hgb A1c^(####) X Brain MRI X X X  X** History,Physical, and KPS X X X X X X X X X X X X (history only) Research BloodSamples X X X X X X  X** 8 × 10 cc green top tubes and one red top tubeMedication Diary ←------------------------------------------→ AdverseEvent report ←------------------------------------------→ ^(#)poly-ICLC(20 μg/kg i.m. and up to 1640 μg) may be administered on the day of thevaccine and on day 4 after each vaccine. ^(##)Includes electrolytes,creatinine, blood urea nitrogen, glucose, AST, ALT, Alk phos, totalbilirubin, LDH, calcium and albumin ^(###)Testing required for patientswith past or current cardiac disease, including symptoms ^(####)Testingonly required for patients with diabetes. <* Additional Therapy>Subjects may undergo additional vaccinations for up to 2 years afteradministration of the first vaccine (See section 4.2.2), if progressionfree status based on the MRI, lack of RLT and anti-GAA immune responseare observed following the initial 8 vaccinations. The additionalvaccines (and poly-ICLC on the day of and on day 4 after each vaccine)may be given every 12 weeks, beginning any week between weeks 34-40.Additional vaccinations may be terminated if tumor-progression, RLT ornegative GAA-response in two consecutive time points is observed.Physical and neurological exam, blood tests to check blood counts andblood chemistry and PBMC samples may be obtained every 12 weeks (at thesame visits for vaccine administrations). An MRI scan may be done atWeeks 12 and 24 to check your tumor response (whether or not your tumoris responding to the vaccines and injections). **For patients whoundergo additional vaccines, head MRI and blood sample collection forimmunological monitoring may be performed every 12 weeks starting at thefirst additional vaccination.

7.8.9 Measurement of Effect

7.8.9.1 Objectives

7.8.9.1.1 Immunogenicity

The response rate and magnitude of CD8+ T-cell responses against theGAA-peptides in post-vaccine PBMC may be assessed using IFN-γ-ELISPOT,and tetramer analysis by flow cytometry as the secondary assay.

ELISPOT assays indicate functional status of the antigen-specific Tcells as cytokine-expression. Flow cytometric analyses using tetramersallow for a relatively accurate estimation of frequency ofantigen-binding T-cells without a major in vitro manipulation of thepatient-derived PBMC, and phenotype analyses, such as the homingreceptor (integrins) expression on antigen-specific T cells.

The biological assays to measure the response in peripheral blood may becarried out at the same time point to avoid inter-assay variability.

Using flow-cytometry, the numbers of lymphocyte subsets such as CD4+ Tcells, CD4+/Foxp3+ regulatory T cells also may be evaluated. Inaddition, in patients who undergo surgical debulking of the progressingtumor, if the tumor tissue is available, infiltration ofantigen-specific CTLs may be evaluated by flow cytometry oftumor-infiltrating lymphocytes with epitope-specific MHC-tetramers.

7.8.9.1.2 Safety

The safety of the administration of the four HLA-A2-restrictedglioma-associated antigen (GAA) epitope-peptides in conjunction with aclass II MHC-restricted Tetanus Toxoid (TT)-derived helper T cellepitope and i.m. poly-ICLC in patients with recurrent grade II gliomasmay be determined.

Endpoints may include incidence and severity of adverse events, usingstandard criteria as well as close clinical follow-up as would beperformed normally in this group of patients following vaccinations. Theregimen may be considered unacceptably toxic if >33% of patients in agiven cohort develop RLT.

7.8.9.1.3 Response and Progression-Free Survival

Tumor recurrence may be assessed minimally at weeks 12 and 24, and every3 months thereafter using MRI scans with contrast enhancement. Sincelow-grade gliomas are infiltrative tumors which typically do not enhancewith contrast administration, for evaluation of response andprogression-free survival, the tumor (i.e., target lesion) may bemeasured from the T2 or FLAIR MM images. In case there is an enhancinglesion at the baseline, careful discussion may be made as to whether thepathology information as WHO grade II tumor truly represents the statusof the tumor. If the enhancing tumor is still considered to be grade II,the size of the enhancing lesion may be used for evaluation. Inaddition, as noted below, emergence of enhancement in previouslynon-enhancing tumor is considered to be progressive disease (PD).

(A) Response (According to RECIST Criteria)

Complete Response (CR): Disappearance of all target lesions.

Partial Response (PR): At least a 30% decrease in the sum of the longestdiameter (LD) of target lesions, taking as reference the baseline sumLD.

Progressive Disease (PD): At least a 20% increase in the sum of the LDof target lesions, taking as reference the smallest sum LD recordedsince the treatment started or the appearance of contrast enhancement ina previously non-enhancing tumor. Because of the possibility ofpseudo-tumor progression, patients may be placed on low-dose steroidsand reimaged before being declared as having PD.

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR norsufficient increase to qualify for PD, taking as reference the smallestsum LD since the treatment started.

(B) Overall Survival (OS) and Progression-Free Survival (PFS)

PFS is defined as the duration of time from start of treatment to timeof progression or death. All patients may be followed for a minimum of 2years, so that their actual 2-year OS and PFS can be determined.

7.8.9.1.4 Analyses of Tumor Tissues Following Vaccinations

Tumor tissues may not be available from all patients in the study.However, the following aspects may be evaluated in an exploratory mannerin all available tumor tissues obtained pre- and/or post-vaccines: (i)Antigen-loss; (ii) up-regulation of anti-apoptotic molecules; and (iii)immune cell infiltration.

7.8.10 Statistical Considerations

7.8.10.1 Assessment of Immunological Responses

Evaluation of immune response may employ both IFN-γ ELISPOT and tetramerassays.

A responder may be defined as a patient who has responded in eitherIFN-γ ELISPOT or tetramer assays. A cohort may be considered worthy offurther investigation if there are at least 4 responses in the 9subjects. This criterion has the property that if the true response rateis <17%, there is <5% probability to observe 4 or more responses, andthat if the true response rate is >66%, there is <5% probability toobserve 3 or fewer responses.

7.8.10.2 Documentation and Evaluation of Safety

The NCI common terminology criteria for adverse events (AE) (CTCAE 3.0)may be used to evaluate toxicity; toxicity may be considered to be anadverse event that is possibly, probably or definitely related totreatment. The maximum grade of toxicity for each category of interestmay be recorded for each patient and the summary results may betabulated by category and grade.

For safety, the regimen may be considered to be excessively toxic if, atany time, the observed rate of regimen-limiting toxicity (RLT)≥33% andat least 2 RLTs have been observed.

The study design has the following properties: if the true rate of RLTis ≥45%, there is at least 90% probability that accrual will stop; ifthe true RLT rate is ≤9%, there is 90% probability that the accrual willnot stop, and that the regimen may be considered safe.

7.8.10.3 Assessment of Clinical Endpoints

All patients may be followed for a minimum of 2 years, so that theiractual 2-year overall survival (OS), progression-free survival (PFS) andresponse rates can be tabulated as exploratory endpoints. PFS is definedas the time interval from the initiation of therapy to progression,based on serial Mill scans. If appropriate, exploratory analyses mayinvestigate the relationship of immune response to imaging response andOS/PFS (using Fisher's exact test and the log rank test, respectively).

7.8.10.4 Demographic Data

Baseline descriptive statistics on all evaluable patients may beprovided for demographic variables (age, sex, race/ethnicity), Karnofskyperformance status, disease stage and status at the time of enrollment(stable disease, progressive disease), and/or treatment regimenspreviously used.

7.9 Example 9

This example describes a study to evaluate the effects of vaccinationswith HLA-A2-restricted glioma antigen-peptides in combination withpoly-ICLC administration for patients with high-risk WHO grade IIastrocytomas and oligoastrocytomas.

7.9.1 Rationale

This Example describes a study of a vaccination regime that is designedto efficiently induce anti-tumor T-cell responses in patients with “highrisk” WHO grade II astrocytoma and oligoastrocytoma; i.e., patients witha >50% likelihood of progression 5 years following surgery alone orsurgery plus postoperative radiation therapy. The regime described inthe study provided in this Example combines subcutaneous injections ofglioma-associated antigen (GAA)-derived cytotoxic T-lymphocyte (CTL)epitope-peptides with simultaneous intramuscular (i.m.) administrationof poly-ICLC.

Adults with supratentorial LGG have a significant risk (24%) of tumorprogression 2 years following treatment with surgery or surgery followedby RT. For unfavorable subsets of these patients, the 2-year risk ofprogression is 40-50%. The study described in this Example has bothimmunoprophylactic and immunotherapeutic potential to reduce the risk oftumor recurrence, which may translate into improved survival.Therapeutically, the immunotherapy approach may suppress the expansionof indolently growing neoplastic low grade II tumor cells.Prophylactically, the approach may prevent anaplastic transformation,which occurs in about one-half of recurrent LGG. The slower growth rateof LGG (in contrast to malignant gliomas) should allow sufficient timeto repeat multiple immunizations, which may lead to the induction ofhigh levels of GAA-specific immunity. In addition, poly-ICLC has beendemonstrated to enhance the vaccine effects in preclinical brain tumormodels (see, e.g., Zhu et al., J. Transl. Med., 5: 10, 2007), and to besafe in malignant glioma patients (see, e.g., Salazar et al.,Neurosurgery, 38: 1096-1103, 1996). Therefore, we hypothesize that thisform of vaccine in combination with poly-ICLC treatment will inducepotent anti-glioma immune response, and will be safe.

7.9.2 Objectives

This Example describes a vaccine study in adults with either WHO gradeII astrocytoma or oligoastrocytoma. The objectives of this Exampleinclude collection of immunological and safety data that may be used inadditional studies. The patients in the study described in this Examplemay be followed for a minimum of 2 years, so that the actual 2-yearoverall survival (OS), 6-month and 2-year progression-free survival(PFS) rates may be determined in an exploratory manner.

7.9.2.1 Induction of GAA-Specific T-Cell Response

The response rate and magnitude of immune response in post-vaccineperipheral blood mononuclear cells (PBMC) against the GAA-peptides inresponse to this form of vaccine may be determined usingIFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

7.9.2.2 Safety

The incidence and severity of adverse events associated with the vaccineregime may be assessed, with an early stopping rule based on thefrequency of Regimen Limiting Toxicity (RLT).

7.9.2.3 Clinical Response

Radiological response may be determined using the standard WHO responsecriteria. 2-year progression-free survival (PFS) may be evaluated in anexploratory manner, based on serial magnetic resonance imaging (MRI)scans.

7.9.2.4 Tumor Tissues for Biological Correlates

For patients who develop progression, biopsy/resection may beencouraged. Whenever post-vaccine tumor tissues are available, they maybe analyzed for GAA expression status and infiltration of GAA-specificT-cells.

7.9.2.5 Influence of RT on Induction of GAA-Specific Immune Response

The rate and magnitude of GAA-specific immune responses in the twocohorts may be compared using IFN-γ-ELISPOT assays and tetramer assays.

7.9.3 Patient Selection

7.9.3.1 Eligibility Criteria

Pathological criteria—Patients should have documented pathologicaldiagnosis of a supratentorial WHO grade II astrocytoma oroligoastrocytoma.

Patients in this study should be HLA-A2 positive based on flowcytometry.

Patients in this study should have recovered from the toxic effects ofprior therapy: 4 weeks from any investigational agent, 4 weeks fromprior cytotoxic therapy and/or at least two weeks from vincristine, 4weeks from nitrosoureas, 3 weeks from procarbazine administration, and 1week for non-cytotoxic agents, e.g., interferon, tamoxifen, thalidomide,cis-retinoic acid, etc. (radiosensitizer does not count). With regard toprevious radiation therapy (RT), there must be at least 6 months fromthe completion of RT (or radiosurgery).

Twp cohorts of patients should be analyzed, based on whether patientshave received prior RT. Cohort 1: Patients should have undergone surgeryor biopsy alone (no postoperative radiation or chemotherapy) and have abaseline MM scan (within 4 weeks of the first vaccine) that shows stabledisease or regression (no progression from the initial surgery/biopsy).Cohort 2: Patients should have undergone surgery or biopsy and radiationtherapy (RT) (including fractionated external beam radiation therapyand/or stereotactic radiosurgery), which was completed ≥6 months priorto enrollment, and have a baseline MRI scan within 4 weeks prior to thefirst vaccine that shows stable disease or regression.

Patients in this study should be (i) age ≥40 with any extent resection;(ii) age 18-39 with incomplete resection (post-op MRI showing >1 cmresidual disease, based on the maximum dimension of residual T2 or FLAIRabnormality from the edge of the surgical cavity either laterally,antero-posteriorally, or supero-inferiorally) or (iii) age 18-39 withneurosurgeon-defined GTR but the tumor size is ≥4 cm (the maximumpreoperative tumor diameter, based on the axial and/or coronal T2 orFLAIR MR images). All patients should be ≥18 years old.

Patients in this study should have a Karnofsky performance status of >60(Appendix I).

Female patients in this study of child-bearing age should havedocumented negative serum βHCG.

Patients in this study should be free of systemic infection. Patientswith active infections (whether or not they require antibiotic therapy)may be eligible after complete resolution of the infection. Patients onantibiotic therapy should be off antibiotics for at least 7 days beforebeginning treatment.

Patients in this study should have adequate organ function as measuredby white blood count ≥2500/mm³; lymphocytes ≥400/mm³; platelets≥100,000/mm³, hemoglobin ≥10.0 g/dL, AST, ALT, GGT, LDH, alkalinephosphatase within 2.5× upper normal limit, and total bilirubin ≤2.0mg/dL, and serum creatinine within 1.5× upper limit of normal limit.Patients in this study should have coagulation tests and PT and PTTshould be within normal limits.

7.9.3.2 Exclusion Criteria

Patients in this study should be excluded if they have presence ofgliomatosis cerebri, cranial or spinal leptomeningeal metastaticdisease.

Patients in this study should be excluded if they have undergone priorchemotherapy or anti-glioma therapy of any type other than radiationtherapy.

Patients in this study should be excluded if they are undergoingconcurrent treatment or medications including: radiation therapy;chemotherapy; interferon (e.g. Intron-A®); allergy desensitizationinjections; growth factors (e.g. Procrit®, Aranesp®, Neulasta®);interleukins (e.g. Proleukin®); and/or any investigational therapeuticmedication.

Patients in this study should not have had prior autoimmune disordersrequiring cytotoxic or immunosuppressive therapy, or autoimmunedisorders with visceral involvement. Patients in this study with anactive autoimmune disorder requiring these therapies also should beexcluded. Mild arthritis requiring NSAID medications should not beexclusionary.

Patients in this study should be excluded if they have usedimmunosuppressives within four weeks prior to entering the study or ifthey anticipate use of immunosuppressive agents. Dexamethasone, or othercorticosteroid medications, if used peri-operative period and/or duringradiotherapy, should be tapered by patients and discontinued at leastfour weeks before administration of the first vaccine in the study.Topical corticosteroids and Inhaled steroids (e.g.: Advair®, Flovent®,Azmacort®) should be acceptable.

Patients in this study should be excluded if they have another cancerdiagnosis, except that the following diagnoses may be allowed: squamouscell cancer of the skin without known metastasis; basal cell cancer ofthe skin without known metastasis; carcinoma in situ of the breast (DCISor LCIS); carcinoma in situ of the cervix; and/or any cancer withoutdistant metastasis that has been treated successfully, without evidenceof recurrence or metastasis for over 5 years.

Patients in this study should be excluded if they have known addictionto alcohol or illicit drugs.

Because patients with immune deficiency are not expected to respond tothis therapy, HIV-positive patients should be excluded from the study.

7.9.4 Peptide Vaccine

7.9.4.1 Peptides

The following peptides may be included in the vaccine formulation:IL-13Rα2₃₄₅₋₃₅₃ 1A9V (ALPFGFILV; SEQ ID NO:3); EphA2₈₈₃₋₈₉₁ (TLADFDPRV;SEQ ID NO:6); Survivin96-104:M2 (LMLGEFLKL; SEQ ID NO:7); WT1₁₂₆₋₁₃₄:Y1(YMFPNAPYL; SEQ ID NO:8); and Tetanus Toxoid (TetA830)(AQYIKANSKFIGITEL; SEQ ID NO:9).

All peptides may be synthesized and the synthetic peptides may bepurified by HPLC. The identity of the synthetic peptides may beconfirmed by verifying their mass and amino acid sequences by massspectrometry. Each lot of peptide may be evaluated as required by theFDA for identity, purity, sterility and pyrogenicity.

The peptides may be vialed under GMP conditions and saved at −70° C.Stability of lyophilized peptides may be tested annually by massspectroscopy.

7.9.4.2 Other Agents

Montanide ISA-51 (SEPPIC Inc., Fairfield, N.J.) may be used as anadditional agent in the peptide vaccines.

7.9.4.3 Dosage and Preparation

An aqueous solution (500 μL) containing each of four HLA-A2-restrictedGAA peptides (300 μg/peptide) and the tetanus peptide (Peptide-tet; 200μg) may be mixed 1/1 with Montanide ISA-51 to form one water-in-oilemulsion (i.e. the total volume/injection is 1 mL).

7.9.4.4 Administration

Patients in this study may be vaccinated subcutaneously in the right orleft upper arms with intact draining axillary nodes. In case patients donot possess intact axillary lymph nodes as the draining nodes, thevaccines may be administered in the upper thigh on the same side withintact inguinal lymph nodes.

The vaccine may be administered on weeks on Weeks 0, 3, 6, 9, 12, 15, 18and 21.

7.9.5 Poly-ICLC

Poly-ICLC may be prepared and packaged in the GMP facility of Bioserv,Corporation (San Diego, Calif.). Poly-ICLC may be supplied in vialscontaining 1 cc of translucent solution with a concentration of 2 mg percc. Poly-ICLC is stable at room temperature for several days, but may bestored refrigerated at about 40° F.

7.9.5.1 Dosage and Administration

Poly-ICLC may be administered intramuscularly at doses of 20 μg/kg andup to 1640 μg/injection, with two injections on days 0 and 4 followingeach vaccination.

The first course of poly-ICLC administration (20 μg/kg i.m. and up to1640 μg/injection) may be administered on the day of the firstGAA/TT-vaccine and on day 4 after the vaccine. For each of the followingrepeated vaccinations (on Weeks 3, 6, 9, 12, 15, 18 and 21), poly-ICLC(20 μg/kg i.m. and up to 1640 μg/injection) may be administered on theday of the vaccine and on day 4 after the vaccine.

With regard to the injection sites, as poly-ICLC is expected to enhancethe antigen-presentation process in the draining lymph nodes, poly-ICLCshould be administered i.m. within the close vicinity to the previouspeptide-injection site (e.g., less than 3 cm from the center of theprevious peptide injection sites).

Poly-ICLC should be administered intramuscularly (i.m.) using steriletechnique, as supplied from the vial, and in the amount prescribed forthe patient's weight (up to 1640 μg/injection). Vital signs may bemonitored before and for at least 20 minutes after the first treatment.

7.9.6 Treatment Plan

The study described in this Example may employ two cohorts of patientsto assess the immunogenicity, safety and clinical efficacy of theGAA/TT-peptide vaccine and poly-ICLC in HLA-A2+ patients with WHO gradeII astrocytoma or oligo-astrocytoma with poor prognostic factors.Because the peptide vaccine is sequestered locally, and the immuneresponse occurs primarily locally and in the draining lymph nodes, thedose of the vaccine should not need to be scaled up proportionately tothe size (by weight or body surface area) of the recipient, as might bedone for a drug whose effect is related to its distribution in bodyfluid. With regard to the dose of poly-ICLC, a fixed dose (20μg/kg/injection and up to 1640 μg/injection) may be employed, which hasbeen demonstrated to be safe and to induce biological responses inpatients with malignant glioma (see, e.g., Salazar et al., Neurosurgery,38: 1096-1103, 1996).

7.9.6.1 Schedule

Eligible patients in Cohort 1 should have undergone surgery or biopsyalone (no postoperative radiation or chemotherapy) and have a baselineMM scan (within 4 weeks of the first vaccine) that shows stable diseaseor regression (no progression from the initial surgery/biopsy); patientsin Cohort 2 should have completed RT≥6 months prior to enrollment, andhave a baseline MRI scan (within 4 weeks prior to the 1^(st) vaccine)showing stable disease or regression. All patients must havediscontinued dexamethasone or similar corticosteroid at least 4 weeksbefore the first vaccine.

Patients may be treated with subcutaneous injections of GAA/TT-vaccineson Weeks 0, 3, 6, 9, 12, 15, 18 and 21. I.m. poly-ICLC may beadministered (20 μg/kg i.m. and up to 1640 μg) on the day of, and on day4 after each vaccine (e.g. if the vaccine is administered on Thursday,poly-ICLC may be administered on the day of vaccine and the followingMonday). Each vaccine may be administered within 2 hours before or afterthe i.m. poly-ICLC administration.

Patients may be evaluated for any possible adverse event, RLT as well asclinical/radiological responses by clinical visits and MRI scanning.

PBMC obtained before the initial vaccine may be used as the base-linesample. If patients demonstrate any positive response in the twoimmunological assays (ELISPOT or Tetramer without RLT ortumor-progression, these patients may be offered additionalGAA/TT-vaccines (see, e.g., Section 7.9.6.2) starting any time betweenon Week 34-40, and every 3 months thereafter until patients demonstratetumor-progression, loss of immune response or RLT.

7.9.6.2 Additional Therapy

On Weeks 0 (baseline), 12, 15, 18, 21, and 24, patients' PBMC may beevaluated for the presence of GAA-specific T-cell responses against GAApeptides. If such a response is observed for any of the GAA peptides,the patient may undergo additional vaccinations with the GAA(s) thatdemonstrated the persisting response as well as poly-ICLC starting anytime between on Week 34-40, and every 12 weeks thereafter up to 2 yearsfrom the initial vaccination. Additional PBMC samples may be obtainedevery 12 weeks (at the same visits for vaccine administrations) forimmunological monitoring. Additional vaccines may be terminated in anyof the following conditions: 1) tumor progression; 2) RLT; or 3)negative immunological response in two consecutive time points.

7.9.6.3 Dose Modification

7.9.6.3.1 Dose Modification for Poly-ICLC

For any Grade 2 or greater flu-like symptoms, including fever andfatigue, poly-ICLC may be discontinued until symptoms return to Grade 0.If Grade 2 or greater flu-like symptoms occur on the day of avaccination, and if the symptoms do not return to Grade 0 by day 4 afterthe vaccination, the next poly-ICLC administration on day 4 after thevaccination may be skipped. If the patient is symptom-free on day 4(Grade 0), poly-ICLC may be resumed at 50% of the original dose. IfGrade 2 or greater flu-like symptoms occur following the poly-ICLCadministration on day 4 after a vaccination, in the next vaccine cycle,two poly-ICLC administrations (on day 0 and 4 following the vaccines)may be given at 50% of the original dose. Pretreatment withacetaminophen 650-1000 mg or with any NSAID may be given. If furtherdosing is well tolerated, the original dose may be subsequentlyre-instituted.

In the case of hepatic enzyme elevation >4× baseline, or any otherunforeseen intolerable side effects of grade 2 or greater, poly-ICLC maybe discontinued until that toxicity has reduced to Grade 1 or less.Poly-ICLC may then be re-administered at one-half the original dose, andthe patient may be closely observed. If the poly-ICLC cannot bere-initiated in the next vaccine cycle, the patient may be withdrawn forthe RLT.

Patients may remain on the original dose of poly-ICLC for grade 1 or 2hematologic toxicity, or grade 1 non-hematologic toxicity. If at the 50%dose level there is no toxicity for a minimum of 2 weeks, the dose maybe escalated back to the starting dose at the discretion of theinvestigator. Subsequent toxicities, should they occur, may require adose reduction to 50%, and no further escalations may be allowed. Iftoxicity reoccurs at the reduced dose, the patient may be taken offtreatment.

7.9.6.3.2 Dosing Delay for the Peptide Vaccines

In circumstances where poly-ICLC administration is suspended, if theevent is not attributable to the peptides/ISA-51 vaccine, vaccineadministrations may not be suspended. If the event is attributable toboth poly-ICLC and peptide-vaccines, both may be suspended. If anadverse event is considered to be solely due to the peptides/ISA-51vaccines, but not poly-ICLC, the vaccine and poly-ICLC administrationsmay be suspended. In circumstances where assessment of an adverse eventis limited, such as by intercurrent illness, or when laboratory studiesare required to assess for other causes of toxicity, the vaccineschedule may be interrupted for up to 4 weeks. Delay of one vaccineadministration by up to 4 weeks will not be considered a protocolviolation if due to an adverse event, regardless of attribution. If oneor more vaccines is delayed by 4 weeks due to an adverse event,regardless of attribution, treatment should be discontinued.

Patients may be observed for regimen limiting toxicity (RLT) throughoutthe study. The following are considered to be RLTs if they are judgedpossibly, probably or definitely associated with treatment. Should theyoccur, individual patients may be taken off study and no furtherinjections may be given.

≥Grade 2 or more bronchospasm or generalized uticaria(hypersensitivity).

≥Grade 2 or more allergic reaction, such as exfoliative erythroderma,anaphylaxis, or vascular collapse.

≥Grade 2 or more autoimmune disease (e.g. hypothyroidism, autoimmuneencephalitis).

Any ≥Grade 3 toxicity possibly, probably, or definitely related to thevaccine with particular attention to the following events.

≥Grade 3 injection site reaction due to peptide-vaccine or poly-ICLCadministration.

≥Grade 3 hematological or hepatic toxicity.

≥Grade 3 neurotoxicity: signs and symptoms that may indicate eithertumor progression or an inflammatory immune response (i.e., pseudo-tumorprogression) that requires a biopsy or resection with pathologicfindings of inflammatory/lymphocytic infiltration.

≥Grade 3 nausea and vomiting without sufficient antiemetic prophylaxisare not considered as RLTs.

Dosing delays >4 weeks for either poly-ICLC or peptide vaccines.

Therapy may be discontinued for the following reasons: (i) Regimenlimiting toxicity—as defined above; (ii) disease progression—at least a20% increase in the sum of the longest diameter of target lesion or theappearance of contrast enhancement in a previously non-enhancing tumor.However, if pseudo-tumor progression is suspected, then the patient maybe placed on dexamethasone, up to 4 mg/day, and reimaged 4-8 weekslater. If they require >4 mg/day dexamethasone, or if their repeatimaging study continues to meet the criteria for disease progression,the patient may be taken off study and further study treatment may bediscontinued. However, if their steroid dose is <4 mg/day and if theirrepeat imaging does not meet the criteria for disease progression, thenthe patient may continue in the study and receive study treatment asprescribed herein. Any cases of suspected tumor progression orpseudo-tumor progression should be reviewed to determine whether thesubject should remain in the study. (iii) Intercurrent illness thatprevents further administration of the vaccine or poly-ICLCadministration. (iv) Pregnancy: Pregnant patients will continue to befollowed for the duration of the pregnancy.

7.9.6.4 Treatment Duration

In the absence of treatment delays due to adverse event(s), treatmentmay continue for 21 weeks (8 vaccinations) or until one of the followingcriteria applies: Regimen Limiting Toxicity (RLT); disease progression;and/or intercurrent illness that prevents further administration oftreatment.

7.9.6.5 Concomitant Treatment

7.9.6.5.1 Acceptable

For fever, acetaminophen may be utilized (325 mg tabs, 1 or 2 p.o. every4 hours). Pre-treatment of patients with acetaminophen may be institutedas warranted by side effects of poly-ICLC. Fevers lasting more than 8hours after treatment may be evaluated in terms of potential infection.

For mild local pain, oral opiates may be planned (oxycodone, 5-10 mgp.o. every 3-4 hours). Pain that is of more than mild-moderate grade maybe investigated for sources other than the therapy, and managedaccordingly.

Dexamethasone (or similar corticosteroid medications) should not be usedfor at least 4 weeks prior the initiation of the vaccine/poly-ICLCtherapy (Week 0). Dexamethasone (up to 4 mg/day) may be used in thesetting of pseudo-tumor progression, and tapered/discontinued as soon aspossible.

Anti-seizure medications should be used as indicated.

Antiemetics, if necessary, may be administered.

Other acceptable medications may include: Topical corticosteroids;nonsteroidal anti-inflammatory agents; anti-histamines (e.g. Claritin®,Allegra®); chronic medications except those listed in Section 7.8.6.5.2;Influenza vaccines (these should be administered at least two weeksprior to the initiation of the study vaccines or at least two weeksafter the 8^(th) (last) vaccine); and/or corticosteroid medicationsadministered parenterally or by inhalation (e.g.: Advair®, Flovent®,Azmacort®).

7.9.6.5.2 Unacceptable

Unacceptable medications may include interferon therapy (e.g.Intron-A®); chemotherapy; allergy desensitization injections; growthfactors (e.g. Procrit®, Aranesp®, Neulasta®); interleukins (e.g.Proleukin®); other investigational medications; and/or illicit drugs.

7.9.7 Correlative/Special Studies

7.9.7.1 Immunological Monitoring

7.9.7.1.1 ELISPOT Assays

Frequencies of glioma associated antigens (GAA)-responsive T-lymphocyteprecursors in peripheral blood mononuclear cells (PBMC) prior to andafter, administration of the GAA-peptide based vaccine may be measuredby ELISPOT assay. The biological responses measured by ELISPOT may bedone at the same time point at least for one individual patient to avoidinter-assay variability. Successful vaccination stimulates clonalpopulations of T cells that are capable of secreting cytokines in anantigen-specific, MHC-restricted fashion. The ELISPOT assay may beutilized to evaluate GAA-specific immune responses of CD8+ T-cellpopulations as well as CD4+ T cells that react against the helper TTpeptide. IFN-γ production may then be evaluated to assess Type-1 T-cellresponse.

A subject may be considered to have responded, if at any of twoconsecutive post-vaccine time points against the same antigen[s] (Weeks12, 15, 18, 21 and 24), the number of spots is double that at baseline,and there are at least 10 spots/20,000 cells, and if the number of thepost-vaccine spots is at least three times the standard-deviation of thepre-vaccine value. Response can be to any one antigen.

7.9.7.1.2 Tetramer Analysis of GAA-Reactive T Cells in Patient's PBMC

Tetramer analyses allow for evaluation of the presence of GAA-specificCD8⁺ T-cells in peripheral blood with a great sensitivity without invitro re-stimulation of the cells. It is expected, based on previousdata available from patients with malignant glioma, that significant (alog or more) increase in the frequency of peptide-responsive CD8⁺ Tcells may be observed in some, but not all, patients immunized withtumor-antigen based vaccines. In an exploratory manner, these PBMCs maybe also evaluated for surface expression of an integrin receptor verylate antigen (VLA)-4, which has been implicated to confer T-cell homingto CNS tumors (see, e.g., Zhu et al., J. Transl. Med., 5: 10, 2007) andchemokine receptors (e.g. CXCR3 and CCR5). Procedures for tetrameranalysis are well established.

Tetramer assays may be done at baseline and at 5 time points aftervaccinations (Weeks 12, 15, 18, 21 and 24). A single time-point positiveresponse for a peptide to be (1+B) % of all CD8⁺ cells positive bytetramer assay may be defined, where B is the percent positive atbaseline, which is usually less than 0.1%. In analogy to the definitionof ELISPOT response, a patient may be considered to have responded ifhe/she has two consecutive single time-point responses for any peptide.

7.9.7.1.3 Flow Cytometric Analyses of Lymphocyte Subsets

Numbers of CD4+ and CD8+ T cells as well as CD4+/Foxp3+ T regulatorycells at serial time points pre- and post-vaccines may be evaluated.

7.9.7.1.4 Analyses of Autoimmunity in Sera

Banked sera may be evaluated for presence of auto-antibodies.

7.9.7.2 Evaluation of Primary and Recurrent Tumor Tissues

GAA-expression in the patients' available tumor tissues may be evaluated(either pre-vaccine or after progression post-vaccines; or both) byimmunohistochemistry (IHC) and reverse transcriptase-polymerase chainreaction (RT-PCR).

If tumors recur following vaccinations, it may be critical to evaluatehow tumors escape the effects of vaccines. To this end, the followingspecific issues may be evaluated as much as the tissue-availabilityallows: (i) Antigen-loss: IHC and RT-PCR may be used to assess whetherthe recurrent tumors express the targeted GAAs, HLA-A2, and antigenprocessing machinery components, such as transporter associated withantigen processing; (ii) up-regulation of anti-apoptotic molecules:although Survivin may be targeted, other anti-apoptotic molecules may beup-regulated, e.g., cFLIP (cellular FLICE (Fas-associated deathdomain-like IL-1ß-converting enzyme) inhibitory protein); and (iii)immune cell infiltration: one reason tumors may escape a vaccine-inducedimmune response is through the failure of reactive T cells to infiltratethe tumor. To examine this, whenever freshly resected tumor tissues (notfixed or frozen) are available, tumor infiltrating lymphocytes (TILs)may be isolated and their numbers, phenotype, and antigen-specificitymay be characterized using HLA-A2 tetramers for each of GAAs. Usingmulti-color flow-cytometry, the function and viability of tetramer⁺ TILsmay be determined by staining for perforin/IFN-γ and Annexin-V,respectively. Control tissues may include pre-vaccine tumors (ifavailable) and recurrent tumors from patients not in the study. Thesestudies may allow for evaluation of whether vaccine-induced T-cellsefficiently traffic to the brain tumor site and maintain their functionand viability.

7.9.8 Study Parameters

This study may be conducted on an outpatient basis, with patientsscheduled to be evaluated on weeks 0, 3, 6, 9, 12, 15, 18, 21 and 24.After this period, if patients do not receive additional vaccines,patients may be off study, and they may be clinically followed every 2-4months thereafter as usually done for patients with the same tumor type.If patients are found to have progressing tumors, other therapies, suchas chemotherapy or resection, may be offered. If patients receiveadditional vaccines, such additional vaccines may be administered every12 weeks, and clinical, immunological and radiological (MRI) monitoringmay be performed at every visit (q12 weeks) until the patients withdraw.Subjects with a complete response may be retreated with additional twovaccinations, with 12-week intervals, and then followed. Vaccinationsmay be halted for any patients with progressive disease or unacceptabletoxicity at any time during the scheduled vaccinations.

7.9.8.1 Pre-Treatment (Screening and Baseline Data)

The following procedures may be undertaken before treatment proceeds:informed Consent should be obtained before initiation of screening; HLAtyping (flow-cytometric evaluation for HLA-A2 positivity); documentationof diagnosis (pathological); complete history and physical examination(with vital signs and weight), including neurological examinations andperformance status; vaccine sites are to be designated with confirmationof intact draining lymph nodes; demographic information should berecorded; CBC and platelets with differential should be evaluated;PT/PTT should be evaluated; Chemistry should be evaluated, includingelectrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, Alkphos, total bilirubin, LDH, calcium and albumin; GGT, phosphorus, andmagnesium should be evaluated; Blood for in vitro assays should betaken; HGBA1C for patients with diabetes mellitus should be performed;ECG and echo cardiogram should be performed for patients with cardiacsymptoms, history or current disease; urinalysis should be performed;MRI of the brain to evaluate the baseline status of disease should beperformed; and/or women of child-bearing potential should beadministered a serum beta-HCG pregnancy test.

7.9.8.2 Evaluation During Treatment

The following procedures may be undertaken as treatment proceeds.Pre-Administration (Weeks 0, 3, 6, 9, 12, 15, 18 and 21 before vaccineadministration on the day of vaccination): history and physicalincluding vital signs, weight, Karnofsky performance status andneurological function; blood for in vitro assays should be taken;Chemistry should be evaluated, including electrolytes, creatinine, bloodurea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin (Except for Week 0); AED levels should be evaluatedif clinically indicated; patients should be screened for adverse eventsfrom previous doses, to include neurological evaluation and skinexamination (injection sites); and/or MRI should be performed (only onWeek 12 among these vaccine injection visits).

Following vaccine administration, all patients should be closelyobserved for adverse events for at least 20 minutes following eachadministration of GAA-peptide vaccine. On the same day, poly-ICLC (i.m.20 mg/kg) may be administered within 2 hours before or after thevaccine, and monitored at least 20 minutes after the poly-ICLCinjection.

7.9.8.3 Week 24 (Post 8 Vaccinations) Evaluation

After the vaccination cycle is complete, the following procedures may beundertaken: history and physical including vital signs, weight,Karnofsky performance status and neurological function; Blood for invitro assays should be taken (Except for Week 3, 6 and 9); CBC andplatelets with differential should be evaluated (Except for Week 0);Chemistry should be evaluated, including electrolytes, creatinine, bloodurea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin (Except for Week 0); AED levels should be evaluatedif clinically indicated; patients should be screened for adverse eventsfrom previous doses, to include neurological evaluation and skinexamination (injection sites); and/or Mill should be performed.

7.9.8.4 Evaluation with Additional Vaccines (Cases with AdditionalVaccines)

Prior to administration with additional vaccines, the followingprocedures may be undertaken: history and physical including vitalsigns, weight, Karnofsky performance status and neurological function;Blood for in vitro assays should be taken (Except for Week 3, 6 and 9);CBC and platelets with differential should be evaluated (Except for Week0); Chemistry should be evaluated, including electrolytes, creatinine,blood urea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin (Except for Week 0); AED levels should be evaluatedif clinically indicated; patients should be screened for adverse eventsfrom previous doses, to include neurological evaluation and skinexamination (injection sites); and/or Mill should be performed.

Following administration with additional vaccines, all patients shouldbe closely observed for adverse events for at least 20 minutes followingeach vaccination. Additional vaccines may be terminated in any of thefollowing conditions: 1) tumor progression; 2) RLT; or 3) negativeimmunological response in two consecutive time points after initiationof additional vaccines.

TABLE 7 Study Calendar Pre-Vaccination Every 12 Consent/ Wks forManagement Table HLA-typing/ Within Treatment (Week) additional Studies& Tests pathology 4 Wks 0 3 6 9 12 15 18 21 24 vaccines* Informedconsent for HLA-typing X HLA-typing X Informed consent for treatmentX-------------------X (if HLA-A2 is positive) Pathology review XVaccination^(#) X X X X X X X X X CBC and Platelets with X X X X X X X XX X differential Coagulation tests (PT and PTT) X Chemistry^(##) X X X XX X X X X X GGT, Phosphorus, Magnesium X AED if clinically indicated X XX X X X X X X Demographics X Concurrent Medications X←------------------------------------------→ Urinalysis X β-HCG X (womenof childbearing potential) EKG or Echocardiogram^(###) X (if clinicallyindicated) Hgb A1c^(####) X Brain MRI X X X  X** History, Physical andKPS X (history X X X X X X X X X X X only) Research Blood Samples X X XX X X  X** 8 × 10 cc green top tubes and one red top tube MedicationDiary ←------------------------------------------→ Adverse Event report←------------------------------------------→ ^(#)Poly-ICLC (20 μg/kgi.m. and up to 1640 μg) may be administered on the day of the vaccineand on day 4 after each vaccine. ^(##)includes electrolytes, creatinine,blood urea nitrogen, glucose, AST, ALT, Alk phos, total bilirubin, LDH,calcium and albumin ^(###)Testing required for participants with past orcurrent cardiac disease, including symptoms. ^(####)Testing onlyrequired for participants with diabetes. *Additional Therapy> Subjectsmay undergo additional vaccinations for up to 2 years afteradministration of the first vaccine (See section 4.2.2), if progressionfree status based on the MRI, lack of RLT and anti-GAA immune responseare observed following the initial 8 vaccinations. The additionalvaccines (and poly-ICLC on the day of and on day 4 after each vaccine)may be given every 12 weeks, beginning any week between weeks 34-40.Additional vaccinations may be terminated if tumor-progression, RLT ornegative GAA-response in two consecutive time points is observed.Physical and neurological exam, blood tests to check blood counts andblood chemistry and PBMC samples may be obtained every 12 weeks (at thesame visits for vaccine administrations). A MRI scan may be done atWeeks 12 and 24 to check your tumor response (whether or not your tumoris responding to the vaccines and injections). **For participants whoundergo additional vaccines, head MRI and blood sample collection forimmunological monitoring may be performed every 12 weeks starting at thefirst additional vaccination.

7.9.9 Measurement of Effect

7.9.9.1 Objectives

7.9.9.1.1 Immunogenicity

The response rate and magnitude of CD8+ T-cell responses against theGAA-peptides in post-vaccine PBMC may be assessed using IFN-γ-ELISPOT,and tetramer analysis by flow cytometry as the secondary assay.

ELISPOT assays indicate functional status of the antigen-specific Tcells as cytokine-expression. Flow cytometric analyses using tetramersallow for a relatively accurate estimation of frequency ofantigen-binding T-cells without a major in vitro manipulation of thepatient-derived PBMC, and phenotype analyses, such as the homingreceptor (integrins) expression on antigen-specific T cells.

The biological assays to measure the response in peripheral blood may becarried out at the same time point to avoid inter-assay variability.

Using flow-cytometry, the numbers of lymphocyte subsets such as CD4+ Tcells, CD4+/Foxp3+ regulatory T cells also may be evaluated. Inaddition, in patients who undergo surgical debulking of the progressingtumor, if the tumor tissue is available, infiltration ofantigen-specific CTLs may be evaluated by flow cytometry oftumor-infiltrating lymphocytes with epitope-specific MHC-tetramers.

7.9.9.1.2 Safety

The safety of the administration of the four HLA-A2-restrictedglioma-associated antigen (GAA) epitope-peptides in conjunction with aclass II MHC-restricted Tetanus Toxoid (TT)-derived helper T cellepitope and i.m. poly-ICLC in patients with grade II astrocytoma andoligoastrocytoma may be determined.

Endpoints may include incidence and severity of adverse events, usingstandard criteria as well as close clinical follow-up as would beperformed normally in this group of patients following vaccinations. Theregimen may be considered unacceptably toxic if >33% of patients in agiven cohort develop RLT.

7.9.9.1.3 Response and Progression-Free Survival

Tumor recurrence may be assessed minimally at weeks 12 and 24, and every3 months thereafter using MRI scans with contrast enhancement. Sincelow-grade gliomas are infiltrative tumors which typically do not enhancewith contrast administration, for evaluation of response andprogression-free survival, the tumor (i.e., target lesion) may bemeasured from the T2 or FLAIR MM images. In case there is an enhancinglesion at the baseline, careful discussion may be made as to whether thepathology information as WHO grade II tumor truly represents the statusof the tumor. If the enhancing tumor is still considered to be grade II,the size of the enhancing lesion may be used for evaluation. Inaddition, as noted below, emergence of enhancement in previouslynon-enhancing tumor is considered to be progressive disease (PD).

(A) Response (According to RECIST Criteria)

Complete Response (CR): Disappearance of all target lesions.

Partial Response (PR): At least a 30% decrease in the sum of the longestdiameter (LD) of target lesions, taking as reference the baseline sumLD.

Progressive Disease (PD): At least a 20% increase in the sum of the LDof target lesions, taking as reference the smallest sum LD recordedsince the treatment started or the appearance of contrast enhancement ina previously non-enhancing tumor. Because of the possibility ofpseudo-tumor progression, patients may be placed on low-dose steroidsand reimaged before being declared as having PD.

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR norsufficient increase to qualify for PD, taking as reference the smallestsum LD since the treatment started.

(B) Overall Survival (OS) and Progression-Free Survival (PFS)

PFS is defined as the duration of time from start of treatment to timeof progression or death. All patients may be followed for a minimum of 2years, so that their actual 2-year OS and PFS can be determined.

7.9.9.1.4 Analyses of Tumor Tissues Following Vaccinations

Tumor tissues may not be available from all patients in the study.However, the following aspects may be evaluated in an exploratory mannerin all available tumor tissues obtained pre- and/or post-vaccines: (i)Antigen-loss; (ii) up-regulation of anti-apoptotic molecules; and (iii)immune cell infiltration.

7.9.10 Statistical Considerations

7.9.10.1 Assessment of Immunological Responses

Evaluation of immune response may employ both IFN-γ ELISPOT and tetramerassays.

A responder may be defined as a patient who has responded in eitherIFN-γ ELISPOT or tetramer assays. A cohort may be considered worthy offurther investigation if there are at least 4 responses in the 9subjects. This criterion has the property that if the true response rateis <17%, there is <5% probability to observe 4 or more responses, andthat if the true response rate is >66%, there is <5% probability toobserve 3 or fewer responses.

7.9.10.2 Documentation and Evaluation of Safety

The NCI common terminology criteria for adverse events (AE) (CTCAE 3.0)may be used to evaluate toxicity; toxicity may be considered to be anadverse event that is possibly, probably or definitely related totreatment. The maximum grade of toxicity for each category of interestmay be recorded for each patient and the summary results may betabulated by category and grade.

For safety, the regimen may be considered to be excessively toxic if, atany time, the observed rate of regimen-limiting toxicity (RLT)≥33% andat least 2 RLTs have been observed.

The study design has the following properties: if the true rate of RLTis ≥45%, there is at least 90% probability that accrual will stop; ifthe true RLT rate is ≤9%, there is 90% probability that the accrual willnot stop, and that the regimen may be considered safe.

7.9.10.3 Assessment of Clinical Endpoints

All patients may be followed for a minimum of 2 years, so that theiractual 2-year overall survival (OS), progression-free survival (PFS) andresponse rates can be tabulated as exploratory endpoints. PFS is definedas the time interval from a patient's pathological diagnosis of WHOgrade II astrocytoma or oligoastrocytoma to progression, based on serialMRI scans. If appropriate, exploratory analyses may investigate therelationship of immune response to imaging response and OS/PFS (usingFisher's exact test and the log rank test, respectively).

7.9.10.4 Demographic Data

Baseline descriptive statistics on all evaluable patients may beprovided for demographic variables (age, sex, race/ethnicity), Karnofskyperformance status, disease stage and status at the time of enrollment(stable disease, progressive disease), and/or treatment regimenspreviously used.

7.10 Example 10

This example describes a study to Evaluate the Effects of vaccinationswith HLA-A2-restricted glioma antigen-peptides in combination withpoly-ICLC for children with newly diagnosed malignant or intrinsic brainstem gliomas (BSG) or incompletely resected non-brainstem high-gradegliomas (HGG) or recurrent unresectable low-grade gliomas (LGG).

7.10.1 Rationale

Currently, there are no effective therapeutic modalities for pediatricmalignant gliomas. Immunotherapy, particularly active vaccinations, hasthe potential to develop as an effective and safe modality. Vaccinesusing GAA-specific peptides, in comparison to whole glioma-derivedantigens, may be more feasible because these vaccines may induceglioma-specific immune responses without theoretical concerns ofauto-immune encephalitis. Evidence from recent studies suggests thatpediatric gliomas and intrinsic brain stem gliomas have a similarpattern of expression of glioma-associated antigens (GAAs), which can betargeted by vaccine-based therapy. In view of the dismal prognosis forchildren with intrinsic brainstem gliomas, incompletely resectedmalignant gliomas, it is appropriate to evaluate the activity and safetyof immunization following radiation therapy in these tumors. Likewise,deep-seated low-grade gliomas also express a similar spectrum of GAAs.Because these lesions commonly become refractory to conventionaltherapy, with increasing morbidity and mortality, it is appropriate toevaluate the potential efficacy of vaccine therapy in those patients whohave had disease progression following at least two chemotherapy orbiological therapy regimens or irradiation.

Administration of poly-ICLC along with the GAA peptides remarkablyenhance the induction of anti-GAA CTL responses and trafficking ofantigen-specific T cells to the brain tumor sites. In the studydescribed in this example, pediatric patients with newly diagnosedmalignant glioma or treatment-refractory low-grade gliomas may bevaccinated with multiple novel GAA-derived HLA-A2 restricted CTLepitopes in combination with intramuscular administration of poly-ICLC.

7.10.2 Objectives

This Example describes a vaccine study in children with newly diagnosedmalignant or intrinsic brain stem gliomas (BSG) or incompletely resectednon-brainstem high-grade gliomas (HGG) or recurrent unresectablelow-grade gliomas (LGG).

7.10.2.1 Induction of GAA-Specific T-Cell Response

The response rate and magnitude of immune response in post-vaccineperipheral blood mononuclear cells (PBMC) against the GAA-peptides inresponse to this form of vaccine may be determined usingIFN-γ-enzyme-linked immuno-spot (ELISPOT) and tetramer assays.

7.10.2.2 Safety

The incidence and severity of adverse events associated with the vaccineregime may be assessed, with an early stopping rule based on thefrequency of Regimen Limiting Toxicity (RLT) in children with newlydiagnosed malignant brain stem gliomas (BSG), and in children with newlydiagnosed incompletely resected non-brain stem malignant gliomas (HGG).The incidence and severity of adverse events associated with the vaccineregimen may also be assessed in patients with treatment-refractory,unresectable low-grade gliomas that have progressed after twochemotherapy or biological therapy regimens or irradiation.

7.10.2.3 Clinical Response

Radiological response may be determined using the standard WHO responsecriteria. 2-year progression-free survival (PFS) may be evaluated in anexploratory manner, based on serial magnetic resonance imaging (MRI)scans.

7.10.2.4 Tumor Tissues for Biological Correlates

For patients with non-brainstem tumors who develop progression,biopsy/tumor debulking may be encouraged. Whenever post-vaccine tumortissues are available, they may be analyzed for GAA expression statusand infiltration of GAA-specific T-cells.

7.10.3 Patient Selection

7.10.3.1 Eligibility Criteria

Pathological criteria—Patients will have glioma. In some embodiments,the glioma patient is in one of the following strata: (i) Stratum A:Newly diagnosed diffuse intrinsic pontine gliomas or any biopsy provenhigh-grade glioma involving the brainstem; (ii) Stratum B: newlydiagnosed, incompletely resected, non-brainstem high-grade glioma (i.e.definite residual tumor visible on imaging); or (iii) Stratum C:Unresectable, progressive low-grade glioma of any subtype that hasrecurred despite two prior chemotherapy or biological therapy regimensand/or radiation therapy; (iv) Stratum D: Newly diagnosed diffuseintrinsic pontine gliomas (DIPG) OR any biopsy proven high-grade glioma*involving the brainstem treated with radiation therapy with or withoutchemotherapy during irradiation; (v) Stratum E: Newly diagnosednon-brainstem high-grade gliomas* (HGG) treated with radiation therapywith or without chemotherapy during irradiation; (vi) Stratum F:Recurrent non-brainstem high-grade gliomas* that have recurred followingtreatment. Patients must have recovered from the toxic effects of priortherapy. Eligible histologies for high-grade glioma include glioblastoma(GBM), anaplastic astrocytoma (AA) or gliosarcoma. Patients with anyoligodendroglioma component may not be eligible for the particularprotocol described in this example.

Patients in this study should be HLA-A2 positive based on flowcytometry.

Patients in Stratum A and B should have received standard involved fieldRT defined as fractionated external beam radiotherapy with total dosesbetween 5000-6000 cGy. Patients in these strata should be registeredwithin 4-12 weeks of completing RT.

Patients in this study should be clinically stable and off or onlow-dose (no more than 0.1 mg/kg/day, max 4 mg/day Dexamethasone)corticosteroid for at least one week prior to study registration.

Patients in this study should be ≥3 and <21 years of age at the time ofstudy.

Patients in this study should have a performance status of ≥50;(Karnofsky if >16 years and Lansky if <16 years of age).

Female patients in this study who are post-menarchal should havedocumented negative serum βHCG.

Patients in this study should be free of systemic infection. Patients onantibiotic therapy should be off antibiotics for at least 7 days beforebeginning treatment.

Patients in this study should have adequate organ function as measuredby: (i) Bone marrow: ANC>1,000/μl; Platelets >100,000/μl (transfusionindependent); Hemoglobin >8 g/dl (may be transfused); (ii) Hepatic:bilirubin ≤1.5× institutional normal for age; SGPT (ALT)<3×institutional normal and albumin ≥2 g/dl; (iii) Renal: Serum creatininebased on age or Creatinine clearance or radioisotope GFR≥70 ml/min/1.73m² Patients in this study should have coagulation tests and PT and PTTwithin normal limits for their age.

Patients in this study should have no overt cardiac, gastrointestinal,pulmonary or psychiatric disease.

For patients in stratum C, recovery from the effects of priorchemotherapy may be required.

7.10.3.2 Exclusion Criteria

Patients in Stratum A and Stratum B of this study should be excluded ifthey have presence of leptomeningeal metastatic disease.

Patients in this study should be excluded if they have gross totallyresected tumors, i.e. no definite visible residual disease on MRI scanat the time of study.

Patients in Stratum A and Stratum B of this study should be excluded ifthey have received any prior chemotherapy or anti-glioma therapy of anytype other than radiation therapy. (Patients in stratum C of this studyshould have received at least two prior chemotherapy or biologic therapyregimens and/or radiation therapy.)

Patients in this study should be excluded if they are undergoingconcurrent treatment or medications including: radiation therapy;interferon (e.g. Intron-A®); allergy desensitization injections; inhaledsteroids (e.g.: Advair®, Flovent®, Azmacort®); growth factors (e.g.Procrit®, Aranesp®, Neulasta®); interleukins (e.g. Proleukin®); and/orany investigational therapeutic medication.

Patients in this study should not have had prior autoimmune disordersrequiring cytotoxic or immunosuppressive therapy, or autoimmunedisorders with visceral involvement. Mild arthritis requiring NSAIDmedications should not be exclusionary.

Patients in this study should be excluded if they have usedimmunosuppressives within four weeks prior to entering the study or ifthey anticipate use of immunosuppressive agents. Dexamethasone, or othercorticosteroid medications, if used peri-operative period and/or duringradiotherapy, should be tapered by patients (no more than 0.1 mg/kg/day,max 4 mg/day dexamethasone) for at least one week before studyregistration. Topical corticosteroids should be acceptable.

Patients in this study should be excluded if they have known addictionto alcohol or illicit drugs.

Because patients with immune deficiency are not expected to respond tothis therapy, HIV-positive patients should be excluded from the study.

7.10.4 Peptide Vaccine

7.10.4.1 Peptides

The following peptides may be included in the vaccine formulation:IL-13Rα2₃₄₅₋₃₅₃ 1A9V (ALPFGFILV; SEQ ID NO:3); EphA2₈₈₃₋₈₉₁ (TLADFDPRV;SEQ ID NO:6); Survivin96-104:M2 (LMLGEFLKL; SEQ ID NO:7); and TetanusToxoid (TetA830) (AQYIKANSKFIGITEL; SEQ ID NO:9).

All peptides may be synthesized and the synthetic peptides may bepurified by HPLC. The identity of the synthetic peptides may beconfirmed by verifying their mass and amino acid sequences by massspectrometry. Each lot of peptide may be evaluated as required by theFDA for identity, purity, sterility and pyrogenicity.

The peptides may be vialed under GMP conditions and saved at −70° C.Stability of lyophilized peptides may be tested annually by massspectroscopy.

7.10.4.2 Other Agents

Montanide ISA-51 (SEPPIC Inc., Fairfield, N.J.) may be used as anadditional agent in the peptide vaccines.

7.10.4.3 Dosage and Preparation

An aqueous solution (400 μL) containing each of four HLA-A2-restrictedGAA peptides (300 μg/peptide) and the tetanus peptide (Peptide-tet; 200μg) may be mixed 1/1 with Montanide ISA-51 to form one water-in-oilemulsion (i.e. the total volume/injection is 800

7.10.4.4 Administration

Patients in this study may be vaccinated subcutaneously in the upper armor thigh.

The vaccine may be administered Q3Wk starting 4-12 wks following thecompletion of RT (Wk 1).

7.10.5 Poly-ICLC

Poly-ICLC may be prepared and packaged in the GMP facility of Bioserv,Corporation (San Diego, Calif.). Poly-ICLC may be supplied in vialscontaining 1 cc of translucent solution with a concentration of 2 mg percc. Poly-ICLC is stable at room temperature for several days, but may bestored refrigerated at about 40° F.

7.10.5.1 Dosage and Administration

The first course of poly-ICLC administration (30 μg/kg i.m.) may beadministered on the day of the first GAA/TT-vaccine. For each of thefollowing repeated vaccinations (Q3W), poly-ICLC (30 μg/kg i.m.) may beadministered on the day of the vaccine.

With regard to the injection sites, as poly-ICLC is expected to enhancethe antigen-presentation process in the draining lymph nodes, poly-ICLCshould be administered i.m. within the close vicinity to the previouspeptide-injection site (e.g., less than 3 cm from the center of theprevious peptide injection sites).

Poly-ICLC should be administered intramuscularly (i.m.) using steriletechnique, as supplied from the vial, and in the amount prescribed forthe patient's weight. The poly-ICLC treatments may be administered onthe same day as the vaccine. Vital signs may be monitored before and forat least 20 minutes after the first treatment.

7.10.6 Treatment Plan

The study described in this Example may employ three strata to assessthe immunogenicity, safety and preliminary clinical efficacy of theGAA/TT-peptide vaccine and poly-ICLC in HLA-A2+ children with newlydiagnosed intrinsic brain stem gliomas (BSG) or biopsy proven GBM, AA orgliosarcoma involving the brainstem (Stratum A); or incompletelyresected non-brainstem GBM, AA or gliosarcoma (Stratum B); or recurrentprogressive low grade gliomas (Stratum C).

7.10.6.1 Schedule

Following diagnosis (for Stratum A and B) or after disease progression(for stratum C), treatment according to the study described in thisExample may be discussed with potentially eligible patients. Allpatients in Stratum A and B may receive fractionated external beamradiation therapy (FEBRT). Patients may be assessed for HLA-A2 status.Eligibility screening and the baseline Mill scan and laboratory studiesshould be completed within 2 weeks of registering to participate in thestudy and within 3 weeks of receiving the first vaccine. Patients inStratum A and B should be registered to participate in the study within4-12 weeks following the completion of FEBRT. The timing of studyregistration for these patients will depend on whether the post-RT MRI(typically done at week 4) shows evidence of increased enhancement ormass effect and the patient is clinically symptomatic/worse. If so,study registration will occur when the patient has been clinicallystable/improved and on low dose (0.1 mg/kg/day max 4 mg decadron) or offsteroids×one week.

Patients may be treated with subcutaneous injections of GAA/TT-vaccinesstarting on Week 1 and every 3 weeks thereafter for up to 8 cycles. I.m.poly-ICLC may be administered (30 μg/kg i.m.) on the same day as thevaccine. Each vaccine may be administered just prior to the i.m.poly-ICLC administration. Poly-ICLC should be administered i.m. withinclose vicinity to the previous peptide-injection site (e.g., less than 3cm from the center of the previous peptide injection sites).

Patients may be evaluated for any possible adverse event, RLT as well asclinical/radiological responses by clinical visits and MM scanning.Follow-up Mills may be performed every 9 weeks starting at Week 7 (Weeks7, 16 and 25).

PBMC obtained before the initial vaccine may be used as the base-linesample. On weeks, 7, 16 and 25, PBMC may be obtained as post-vaccinesamples. Immunological assays may be performed for all PBMC samplesobtained from at least one participant at one time, so that inter-assayvariability will be avoided.

7.10.6.2 Additional “Continuation” Therapy

After the scheduled 8^(th) vaccination, if the patient demonstratesradiological response (i.e. complete or partial response) or stabledisease without RLT, the patient may receive additional peptidevaccinations in conjunction with poly-ICLC starting 6 weeks after the8^(th) vaccination, and every 6 weeks thereafter up to 2 years from theinitial vaccination as long as there is no tumor progression and no RLT.Additional PBMC samples may be obtained at the same visits for vaccineadministrations for immunological monitoring. Additional vaccines may beterminated in any of the following conditions: 1) tumor progression; 2)RLT; or 3) patient withdrawal.

7.10.6.3 Dose Modification

7.10.6.3.1 Dose Modification for Poly-ICLC

Pretreatment with acetaminophen or with any NSAID should be given beforeeach poly-ICLC dose. For Grade 2 or greater constitutional symptoms thatpersist for greater than 48 hours after the injection, the nextpoly-ICLC dose should be given at 67% of the original dose (i.e. 20μg/kg). If further dosing is well tolerated, the original dose may besubsequently re-instituted. If grade 2 or greater symptoms again occurdespite one dose reduction and last >48 hours, the patient may bewithdrawn for RLT.

In the case of hepatic enzyme elevation >5× baseline (Grade 3), or anyintolerable grade 2 non-hematologic toxicity that lasts for ≥7 days,poly-ICLC may be held until that toxicity has reduced to Grade 1 orless. Poly-ICLC may then be re-administered at two-thirds of theoriginal dose (i.e. 20 μg/kg), and the participant may be closelyobserved. If the same dose-limiting toxicity again recurs despite thedose reduction, the participant may be withdrawn for RLT.

For grade 3 or greater hematologic toxicity, the next dose should bereduced to 67% (i.e. 20 μg/kg) as long as the toxicity has resolved tograde 1 or less by the time the next dose is due. If the toxicity hasnot resolved by the time the next dose is due, the patient is offtreatment. If the same dose-limiting hematologic toxicities again occurdespite the reduced dose, the patient may be taken off treatment forRLT.

7.10.6.3.2 Dosing Delay for the Peptide Vaccines

In circumstances where poly-ICLC administration is suspended, if theevent is not attributable to the peptides/ISA-51 vaccine, vaccineadministration should continue on schedule. In circumstances whereassessment of an adverse event is limited, such as by intercurrentillness, or when laboratory studies are required to assess for othercauses of toxicity, the vaccine schedule may be interrupted for up to 6weeks. If vaccine administration is delayed by longer than 6 weeks dueto an adverse event other than for pseudo-tumor progression, regardlessof attribution, treatment should be discontinued.

Patients may be observed for regimen limiting toxicity (RLT) throughoutthe study. The following are considered to be RLTs if they are judgedpossibly, probably or definitely associated with treatment. Should theyoccur, individual patients may be taken off study and no furtherinjections may be given.

≥Grade 2 or more bronchospasm or generalized uticaria(hypersensitivity).

≥Grade 2 or more allergic reaction, such as exfoliative erythroderma,anaphylaxis, or vascular collapse.

Any ≥Grade 3 non-hematologic toxicity (excluding hepatic toxicity)possibly, probably, or definitely related to the therapy regimenincluding Grade 3 injection site reaction due to peptide-vaccine orpoly-ICLC administration.

≥Grade 3 hematologic or hepatic toxicity that recurs despite a 33% dosereduction or does not resolve to grade 1 or less by the time the nextdose is due.

Intolerable grade 2 non-hematologic toxicity lasting ≥7 days that recursdespite a 33% dose reduction or does not resolve to grade 1 or less bythe time the next dose is due.

Grade 2 or greater constitutional symptoms that persist for >48 hoursdespite a dose reduction.

≥Grade 3 neurotoxicity due to a regimen-related inflammatory immuneresponse (i.e., pseudo-tumor progression that does not respond to a 7day trial of 0.3 mg/kg day decadron (max 12 mg/day) and/or requiresdebulking surgery, if feasible.

≥Grade 3 nausea and vomiting despite sufficient anti-emetic prophylaxis.

Dosing delays >6 weeks for either poly-ICLC or peptide vaccines due totoxicity other than PTP.

Therapy may be discontinued for the following reasons: (i) Regimenlimiting toxicity other than PTP—as defined above; (ii) diseaseprogression—at least a 25% increase in the product of the longest tumordiameter and its perpendicular diameter on MRI scan. (iii) Intercurrentillness that prevents further administration of the vaccine or poly-ICLCadministration for longer than 6 weeks. (iv) Pregnancy: Pregnantpatients will continue to be followed for the duration of the pregnancy.

7.10.6.4 Treatment Duration

In the absence of treatment delays due to adverse event(s), treatmentmay continue for 25 weeks (8 vaccinations and the follow-up visit atWeek 25) or until one of the Off-Treatment criteria in Section7.10.6.3.2 occurs.

7.10.6.5 Concomitant Treatment

7.10.6.5.1 Acceptable

Patients should receive a dose of acetaminophen (15 mg/kg up to a max of1000 mg) 30-60 minutes before each poly-ICLC administration. For feverfollowing the injection, acetaminophen (15 mg/kg up to a max of 1000 mgq 4-6 hours prn, not to exceed 4 g/day) may be given. Patients withfevers lasting longer than 48 hours should be evaluated for potentialinfection.

For mild local pain, oral opiates may be used (tylenol and codeine 0.5mg/kg p.o. every 4 hours). Pain that is of more than mild-moderate gradewill be investigated for non-therapy related causes, and managedaccordingly.

Dexamethasone—no more than 0.1 mg/kg/day, max 4 mg/day for at least oneweek prior to the initiation of the vaccine/poly-ICLC therapy (Week 0).Dexamethasone dose may be increased in the setting of pseudo-tumorprogression and tapered/discontinued as soon as possible.

Anti-seizure medications should be used as indicated.

Antiemetics, if necessary, may be administered.

Other acceptable medications may include: Topical corticosteroids;nonsteroidal anti-inflammatory agents; anti-histamines (e.g. Claritin®,Allegra®); chronic medications except those listed in Section7.10.6.5.2; and/or Influenza vaccines (these should be administered atleast two weeks prior to the initiation of the study vaccines or atleast two weeks after the 8^(th) (last) vaccine).

7.10.6.5.2 Unacceptable

Unacceptable medications may include interferon therapy (e.g.Intron-A®); chemotherapy; allergy desensitization injections;corticosteroid medications administered parenterally or by inhalation(e.g.: Advair®, Flovent Azmacort®); growth factors (e.g. Procrit®,Aranesp®, Neulasta®); interleukins (e.g. Proleukin®); otherinvestigational medications; and/or illicit drugs.

7.10.7 Correlative/Special Studies

7.10.7.1 Immunological Monitoring

7.10.7.1.1 ELISPOT Assays

Frequencies of glioma associated antigens (GAA)-responsive T-lymphocyteprecursors in peripheral blood mononuclear cells (PBMC) prior to andafter, administration of the GAA-peptide based vaccine may be measuredby ELISPOT assay. The biological responses measured by ELISPOT may bedone at the same time point at least for one individual patient to avoidinter-assay variability. Successful vaccination stimulates clonalpopulations of T cells that are capable of secreting cytokines in anantigen-specific, MHC-restricted fashion. The ELISPOT assay may beutilized to evaluate GAA-specific immune responses of CD8+ T-cellpopulations as well as CD4+ T cells that react against the helper TTpeptide. IFN-γ production may then be evaluated to assess Type-1 T-cellresponse.

A subject may be considered to have responded, if at any of twoconsecutive post-vaccine time points against the same antigen[s] (Weeks12, 15, 18, 21 and 24), the number of spots is double that at baseline,and there are at least 10 spots/20,000 cells, and if the number of thepost-vaccine spots is at least three times the standard-deviation of thepre-vaccine value. Response can be to any one antigen.

7.10.7.1.2 Tetramer Analysis of GAA-Reactive T Cells in Patient's PBMC

Tetramer analyses allow for evaluation of the presence of GAA-specificCD8⁺ T-cells in peripheral blood with a great sensitivity without invitro re-stimulation of the cells. It is expected, based on previousdata available from patients with malignant glioma, that significant (alog or more) increase in the frequency of peptide-responsive CD8⁺ Tcells may be observed in some, but not all, patients immunized withtumor-antigen based vaccines. In an exploratory manner, these PBMCs maybe also evaluated for surface expression of an integrin receptor verylate antigen (VLA)-4, which has been implicated to confer T-cell homingto CNS tumors (see, e.g., Zhu et al., J. Transl. Med., 5: 10, 2007) andchemokine receptors (e.g. CXCR3 and CCR5). Procedures for tetrameranalysis are well established.

Tetramer assays may be done at baseline and at 5 time points aftervaccinations (Weeks 12, 15, 18, 21 and 24). A single time-point positiveresponse for a peptide to be (1+B) % of all CD8⁺ cells positive bytetramer assay may be defined, where B is the percent positive atbaseline, which is usually less than 0.1%. In analogy to the definitionof ELISPOT response, a patient may be considered to have responded ifhe/she has two consecutive single time-point responses for any peptide.

7.10.7.1.3 Flow Cytometric Analyses of Lymphocyte Subsets

Numbers of CD4+ and CD8+ T cells as well as CD4+/Foxp3+ T regulatorycells at serial time points pre- and post-vaccines may be evaluated.

7.10.7.2 Evaluation of Primary and Recurrent Tumor Tissues

GAA-expression in the patients' available tumor tissues may be evaluated(either pre-vaccine or after progression post-vaccines; or both) byimmunohistochemistry (IHC) and reverse transcriptase-polymerase chainreaction (RT-PCR).

If tumors recur following vaccinations, it may be critical to evaluatehow tumors escape the effects of vaccines. To this end, the followingspecific issues may be evaluated as much as the tissue-availabilityallows: (i) Antigen-loss: IHC and RT-PCR may be used to assess whetherthe recurrent tumors express the targeted GAAs, HLA-A2, and antigenprocessing machinery components, such as transporter associated withantigen processing; (ii) up-regulation of anti-apoptotic molecules:although Survivin may be targeted, other anti-apoptotic molecules may beup-regulated, e.g., cFLIP (cellular FLICE (Fas-associated deathdomain-like IL-1ß-converting enzyme) inhibitory protein); and (iii)immune cell infiltration: one reason tumors may escape a vaccine-inducedimmune response is through the failure of reactive T cells to infiltratethe tumor. To examine this, whenever freshly resected tumor tissues (notfixed or frozen) are available, tumor infiltrating lymphocytes (TILs)may be isolated and their numbers, phenotype, and antigen-specificitymay be characterized using HLA-A2 tetramers for each of GAAs. Usingmulti-color flow-cytometry, the function and viability of tetramer⁺ TILsmay be determined by staining for perforin/IFN-γ and Annexin-V,respectively. Control tissues may include pre-vaccine tumors (ifavailable) and recurrent tumors from patients not in the study. Thesestudies may allow for evaluation of whether vaccine-induced T-cellsefficiently traffic to the brain tumor site and maintain their functionand viability.

7.10.8 Study Parameters

This study may be conducted on an outpatient basis, with patientsscheduled to be evaluated every 3 weeks for up to 8 vaccinations. Ifpatients receive additional vaccines, administered every 6 weeks as partof the continuation phase, clinical, immunological and radiological (MM)monitoring may be performed at every visit (Q6Wk) until one of thecriteria for ending treatment are met. Vaccinations may be halted forany patients with progressive disease or unacceptable toxicity at anytime during the scheduled vaccinations.

7.10.8.1 Pre-Treatment (Screening and Baseline Data)

The following procedures may be undertaken before treatment proceeds:informed Consent should be obtained before initiation of screening; HLAtyping (flow-cytometric evaluation for HLA-A2 positivity); anddocumentation of diagnosis (histological for non-brainstem primaries(i.e., Stratum B); pathological or imaging for brainstem primaries);complete history and physical examination (with vital signs and weight),including neurological examinations and performance status; concurrentmedication demographic information should be recorded; CBC and plateletswith differential should be evaluated; PT/PTT should be evaluated;comprehensive metabolic panel should be evaluated, includingelectrolytes, creatinine, blood urea nitrogen, glucose, AST, ALT, Alkphos, total bilirubin, LDH, calcium and albumin; GGT, phosphorus, andmagnesium should be evaluated; Blood for in vitro assays should betaken; urinalysis should be performed; Mill of the brain should beperformed; and/or women of child-bearing potential should beadministered a serum beta-HCG pregnancy test.

7.10.8.2 Evaluation During Treatment

The following procedures may be undertaken as treatment proceeds.Pre-Administration: history and physical including vital signs, weight,performance status, concurrent medication, and neurological function;blood for in vitro assays should be taken; Chemistry should beevaluated, including electrolytes, creatinine, blood urea nitrogen,glucose, AST, ALT, Alk phos, total bilirubin, LDH, calcium and albumin;patients should be screened for adverse events from previous doses, toinclude neurological evaluation and skin examination (injection sites);and/or MM should be performed (every 9 weeks starting Week 6; i.e. Weeks6, 15 and 24).

Following vaccine administration, all patients should be closelyobserved for adverse events for at least 20 minutes following eachadministration of GAA-peptide vaccine. On the same day, poly-ICLC (i.m.30 mg/kg) will be administered after each vaccine, and patients will bemonitored for at least 20 minutes after the poly-ICLC injection.

7.10.8.3 Week 24 (Post 8 Vaccinations) Evaluation

After the vaccination cycle is complete, the following procedures may beundertaken: history and physical including vital signs, weight,performance status, concurrent medication, and neurological function;Blood for in vitro assays should be taken; CBC and platelets withdifferential should be evaluated; Chemistry should be evaluated,including electrolytes, creatinine, blood urea nitrogen, glucose, AST,ALT, Alk phos, total bilirubin, LDH, calcium and albumin (Except forWeek 0); and/or patients should be screened for adverse events fromprevious doses, to include neurological evaluation and skin examination(injection sites).

7.10.8.4 Evaluation with Additional “Continuation” Vaccines

Prior to administration with additional vaccines, the followingprocedures may be undertaken: history and physical including vitalsigns, weight, performance status, concurrent medication, andneurological function; Blood for in vitro assays should be taken; CBCand platelets with differential should be evaluated; Chemistry should beevaluated, including electrolytes, creatinine, blood urea nitrogen,glucose, AST, ALT, Alk phos, total bilirubin, LDH, calcium and albumin(Except for Week 0); patients should be screened for adverse events fromprevious doses, to include neurological evaluation and skin examination(injection sites); and/or Mill should be performed.

Following administration with additional vaccines, all patients shouldbe closely observed for adverse events for at least 20 minutes followingeach vaccination. Additional vaccines may be terminated in any of thefollowing conditions: 1) tumor progression; 2) RLT; or 3) negativeimmunological response in two consecutive time points after initiationof additional vaccines.

TABLE 8 Study Calendar every 12 Wks for Management Table Pre- additionalStudies & Tests vac^(@) 0 3 6 9 12 15 18 21 24 vaccines * Informedconsent X HLA-typing X Pathology review X Vaccination ^(#) X X X X X X XX X (Q6Wks) CBC with differential X X X X X X X X X X X (Q6Wks)Comprehensive chemistry X X X X X X X X X X X (Q6Wks) including LDH, ALTand AST Demographics X Concurrent Medications X←--------------------------------------------------------------→Urinalysis X β-HCG (women of X childbearing potential) Brain MRI X X X XX** History, Physical, and KPS X X X X X X X X X X X (Q6Wks) 35 cc greentop tubes (7 X X X X X** tubes) ^(@)Within 2 weeks of registration (Wk−2 to 0) for H&P, MRI and laboratory tests. HLA-typing is done any timeafter signing Part 1 of the consent form. ^(#) poly-ICLC (30 μg/kg i.m.)may be administered after each vaccine. <* Additional Therapy> Subjectsmay undergo additional vaccinations, if progression free status based onthe MRI and lack of RLT are observed following the initial 8vaccinations. The additional vaccines and poly-ICLC may be given every 6weeks, starting 6 weeks after the 8^(th) vaccination, and up to 2 yearsfrom the initial vaccination.. Additional vaccinations may be terminatedif Off-Treatment criteria are met. **For patients who undergo additionalvaccines, brain MRI and blood sample collection for immunologicalmonitoring may be performed every 6 weeks.

7.10.9 Measurement of Effect

7.10.9.1 Objectives

7.10.9.1.1 Immunogenicity

The response rate and magnitude of CD8+ T-cell responses against theGAA-peptides in post-vaccine PBMC may be assessed using IFN-γ-ELISPOT,and tetramer analysis by flow cytometry as the secondary assay.

ELISPOT assays indicate functional status of the antigen-specific Tcells as cytokine-expression. Flow cytometric analyses using tetramersallow for a relatively accurate estimation of frequency ofantigen-binding T-cells without a major in vitro manipulation of thepatient-derived PBMC, and phenotype analyses, such as the homingreceptor (integrins) expression on antigen-specific T cells.

The biological assays to measure the response in peripheral blood may becarried out at the same time point to avoid inter-assay variability.

Using flow-cytometry, the numbers of lymphocyte subsets such as CD4+ Tcells, CD4+/Foxp3+ regulatory T cells also may be evaluated. Inaddition, in patients who undergo surgical debulking of the progressingtumor, if the tumor tissue is available, infiltration ofantigen-specific CTLs may be evaluated by flow cytometry oftumor-infiltrating lymphocytes with epitope-specific MHC-tetramers.

7.10.9.1.2 Safety

The safety of the administration of the four HLA-A2-restrictedglioma-associated antigen (GAA) epitope-peptides in conjunction with aclass II WIC-restricted Tetanus Toxoid (TT)-derived helper T cellepitope and i.m. poly-ICLC in patients with newly diagnosed brainstemand non-brainstem malignant gliomas immediately following irradiation(Stratum A and B, respectively) and in patients withtreatment-refractory, unresectable, low-grade glioma (Stratum C) may bedetermined.

Endpoints may include incidence and severity of adverse events, usingstandard criteria as well as close clinical follow-up as would beperformed normally in this group of patients following vaccinations. Theregimen may be considered unacceptably toxic if >33% of patients in agiven cohort develop RLT.

7.10.9.1.3 Response and Progression-Free Survival

For evaluation of response and progression-free survival, the tumor(i.e., target lesion) may be measured from gadolinium (Gd)-enhanced T1MRI images or, for tumors with non-enhancing components, fromT2-weighted images.

(A) Response (According to RECIST Criteria)

Complete Response (CR): Complete disappearance on MM of all visibletumor and mass effect, on a stable or decreasing dose of corticosteroids(or only adrenal replacement doses), accompanied by a stable orimproving neurologic examination, and maintained for at least 6 weeks.

Partial Response (PR): Greater than or equal to 50% reduction in tumorsize by bi-dimensional measurement on a stable or decreasing dose ofcorticosteroids, accompanied by a stable or improving neurologicexamination, and maintained for at least 6 weeks.

Progressive Disease (PD): neurologic abnormalities or worseningneurologic status not explained by causes unrelated to tumor progression(e.g., anticonvulsant or corticosteroid toxicity, electrolytedisturbances, sepsis, hyperglycemia, etc.), or a greater than 25%increase in the bi-dimensional measurement, or increasing doses ofcorticosteroids required to maintain stable neurologic status orimaging.

Stable Disease (SD): Neurologic exam is at least stable and maintenancecorticosteroid dose not increased, and MR imaging meets neither thecriteria for PR or the criteria for Progressive Disease. If thiscategory is to be reported as of possible clinical benefit, StableDisease status must be maintained for at least 12 weeks.

Pseudo-Progressive Disease (Pseudo-PD) Patients with pseudo-progression,who remain on study and ultimately experience SD, PR, or CR may beclassified as both pseudo-PD and either SD, PR, or CR, respectively, forresponse determinations.

(B) Overall Survival (OS) and Progression-Free Survival (PFS)

PFS is defined as the duration of time from start of treatment to timeof progression or death. All patients will be followed to determine OSand PFS.

7.10.9.1.4 Analyses of Tumor Tissues Following Vaccinations

Tumor tissues may not be available from all patients in the study.However, the following aspects may be evaluated in an exploratory mannerin all available tumor tissues obtained pre- and/or post-vaccines: (i)Antigen-loss; (ii) up-regulation of anti-apoptotic molecules; and (iii)immune cell infiltration.

7.10.10 Statistical Considerations

7.10.10.1 Assessment of Immunological Responses

Evaluation of immune response may employ both IFN-γ ELISPOT and tetramerassays.

A responder may be defined as a patient who has responded in eitherIFN-γ ELISPOT or tetramer assays. Each of the three strata should beevaluated independently. A stratum will be considered worthy of furtherinvestigation if there are at least 5 responses in the 12 subjects. Thiscriterion has the property that if the true response rate is <18%, thereis <5% probability to observe 5 or more responses, and that if the trueresponse rate is >68%, there is <5% probability to observe 4 or fewerresponses.

7.10.10.2 Documentation and Evaluation of Safety

The NCI common terminology criteria for adverse events (AE) (CTCAE 3.0)may be used to evaluate toxicity; toxicity may be considered to be anadverse event that is possibly, probably or definitely related totreatment. The maximum grade of toxicity for each category of interestmay be recorded for each patient and the summary results may betabulated by category and grade.

For safety, the regimen may be considered to be excessively toxic if, atany time, the observed rate of regimen-limiting toxicity (RLT)≥33% andat least 2 RLTs have been observed.

The study design has the following properties: if the true rate of RLTin this patient population is ≥42%, there is at least 90% probabilitythat accrual will stop; if the true RLT rate is ≤8.7%, there is 90%probability that the accrual will not stop, and that the regimen will beconsidered safe.

7.10.10.3 Assessment of Clinical Endpoints

Clinical responses may be documented, and the response rate and its 95%confidence bounds computed. All patients may be followed for assessmentof overall survival (OS) and progression-free survival (PFS). PFS isdefined for Stratum A and B as the time interval from a patient'sdiagnosis to death or progression, and for Stratum C from the time ofstudy registration to death or progression, based on serial MRI scans.If appropriate, exploratory analyses may investigate the relationship ofimmune response to clinical response and OS/PFS (using Fisher's exacttest and the log rank test, respectively).

7.10.10.4 Demographic Data

Baseline descriptive statistics on all evaluable patients may beprovided for demographic variables (age, sex, race/ethnicity), Karnofskyor Lansky performance status, disease stage and status at the time ofenrollment (stable disease, progressive disease), and treatment regimenspreviously used (for Stratum C).

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it may be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

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1-2. (canceled)
 3. A pharmaceutical composition comprising an IL-13Rα2peptide, an EphA2 peptide, YKL-40 peptide, and a GP100 peptide. 4-5.(canceled)
 6. The pharmaceutical composition of claim 3, wherein theIL-13Rα2 peptide comprises any one of SEQ ID NOs:1-4, the EphA2 peptidecomprises SEQ ID NO:6, the YKL-40 peptide comprises SEQ ID NO:10, andthe GP100 peptide comprises SEQ ID NO:11. 7-24. (canceled)
 25. A methodof vaccinating a patient against glioma, wherein a compositioncomprising EphA2₈₈₃₋₈₉₁ is introduced into the patient under conditionssufficient for the patient to develop a CTL response.
 26. (canceled) 27.The pharmaceutical composition of claim 3, wherein one or more of thepeptides are loaded on dendritic cells. 28-29. (canceled)
 30. Thepharmaceutical composition of claim 3, further comprising an adjuvant.31. The pharmaceutical composition of claim 30, wherein the adjuvant isMontanide ISA-51. 32-38. (canceled)
 39. A method for treating,preventing, or managing brain cancer in a subject in need thereofcomprising administering to said subject the pharmaceutical compositionof claim
 3. 40. The method of claim 39, further comprising administeringto the subject a helper T cell epitope.
 41. The method of claim 40,wherein the helper T cell epitope is the PADRE peptide, a Tetanus toxoidpeptide, or the HBV₁₂₈₋₁₄₀ core peptide.
 42. The method of claim 39,further comprising administering to the subject an immune responsemodifier.
 43. The method of claim 42, wherein the immune responsemodifier is poly-ICLC or imiquimod.
 44. The method of claim 39, whereinthe subject is human.
 45. The method of claim 39, wherein thepharmaceutical composition is administered to the subject subcutaneouslyor intra-nodally.
 46. The method of claim 39, wherein the pharmaceuticalcomposition is cell-free.
 47. The method of claim 39, wherein the braincancer is glioblastoma.
 48. The method of claim 39, wherein the subjecthas failed prior therapy with temozolomide, radiation therapy, andsurgery.
 49. The method of claim 39, further comprising administeringbevacizumab to the subject.
 50. The method of claim 39, wherein thepharmaceutical composition comprises 250 to 400 μg of the IL-13Rα2peptide and 250 to 400 μg of the EphA2 peptide.
 51. The method of claim39, wherein one or more of the peptides are loaded on dendritic cells.